This work was supported by the National Institutes of Health through Grant Award Numbers R37-CA260223, R21CA246414. We thank the NCSU flow cytometry core for training and guidance on flow cytometry analysis. Schematics were created with Biorender.com

All the procedures involving human primacy cells and retroviral vectors were performed in compliance with North Carolina State University&#39;s Biological Safety guidelines and approved by the Environmental Health and Safety Office. Human peripheral blood mononuclear cells were purchased as buffy coats from commercial sources. Primary human cells must be isolated from human buffy coat fractions and require Biosafety Level 2 clearance and detailed standard operating procedures and approval from the institution where the work is to take place. Viral vectors, including the retroviral vector supernatants used for the transduction prepared as previously described19, can be classified either as Biosafety Level 1 or Biosafety Level 2 depending on the encoded protein and require approval from the relevant institutional biosafety committee.
1. Making the macroporous alginate scaffolds
<ol>
	<li>Prepare a stock solution of 0.4% (weight per volume) calcium D gluconate by adding sterile-filtered deionized water to calcium D gluconate in a beaker. Stir on medium speed until the calcium gluconate has dissolved (~1.5 h), sterile-filter, and store at room temperature. 
	NOTE: It is not necessary to check the pH of the solution.</li>
	<li>Prepare a solution of 2% (weight per volume) alginate in a screw cap vial or beaker. Carefully add the ultrapure alginate to the vessel and add sterile-filtered deionized water to the alginate. Choose the largest stir bar possible that will not impede mixing and add it to the vessel. 
	NOTE: Information on the type of ultrapure alginate used for this protocol can be found in the Table of Materials and on the Novamatrix website20. Different alginates were not explored for these scaffolds, as they can lead to undesired changes in porosity and biocompatibility of the scaffolds9,10,11.</li>
	<li>Stir the solution on high speed until the alginate dissolves (~1 h). If there are large clumps on the sides of the vessel, tilt the vessel to dislodge them. Small amounts of alginate on the sides will dissolve while stirring and are not cause for concern. 
	NOTE: It is not necessary to check the pH of the solution.</li>
	<li>When the alginate dissolves, reduce the stirring speed, slowly add an equal volume of 0.4% calcium D gluconate to the alginate solution to prevent crosslinking clumps from forming, and stir vigorously for 15 min.</li>
	<li>Tilt the beaker or vial sideways to remove any clumps that may have formed. 
	NOTE: It is recommended to use a homogenizer. The final solution should be optically clear.</li>
	<li>Pipette the desired volume of the solution into each well of a 48-well plate or 24-well plate. Use 300 &#181;L/well for a 48-well plate and 1 mL/well for a 24-well plate. Cast slowly and change tips frequently, as the solution will be viscous and will stick to the tip.</li>
	<li>Cover the plates with lids and freeze overnight at -20 &#730;C.</li>
	<li>Prepare the plate for the lyophilizer by removing the lid and securing the top with wipes and rubber bands. Place the plates in 750 mL to 2,000 mL lyophilizer flasks, and place on the lyophilizer for 72 h.</li>
	<li>After 72 h, remove the plate from the lyophilizer and replace the lid. Seal the plate and store at 4 &#730;C. For long term storage, vacuum-seal the plate before storing at 4 &#730;C. Keep dry until needed. 
	&#8203;NOTE: Make extra calcium-alginate solution due to volume error (some sticks to the walls of the vial, pipette tips). For example, to make four scaffolds in a 24-well plate, 4 mL of combined solution (2 mL alginate + 2 mL calcium D gluconate) is required, but it is recommended to prepare 2.5 mL of 2% alginate solution and 2.5 mL of 0.4% calcium D gluconate solution.</li>
</ol>
2. Transduction
<ol>
	<li>Concentration of viral supernatant using 100 kDa filters
	<ol>
		<li>Hydrate the centrifugal filter for 2 min with 1 mL of 1x phosphate-buffered saline (PBS). Discard the PBS.</li>
		<li>Concentrate the viral suspension by adding 2 mL of viral suspension (1 &#215; 106 TU/mL) and centrifuging it through the centrifugal filter at 1,500 &#215; g for 10 min in a swinging bucket rotor. 
		NOTE: One to two hundred microliters of concentrated virus is needed per scaffold.</li>
		<li>Spin for a few additional minutes if the concentrated virus suspension volume is greater than 200 &#181;L.</li>
		<li>Repeat steps 2.1.1-2.1.3 for each scaffold. 
		NOTE: It is also acceptable to concentrate a large stock of virus and take 100-200 &#181;L aliquots from the concentrated stock.</li>
	</ol>
	</li>
	<li>Seed activated cells and virus together.
	<ol>
		<li>For each scaffold, prepare an aliquot by suspending 1 &#215; 106 activated peripheral blood mononuclear cells (PBMCs) in 50 &#181;L of complete cell culture media (250 mL Click&#39;s media + 250 mL RPMI 1640 media + 50 mL fetal bovine serum + 5 mL glutamine substitute + 5 mL penicillin/streptomycin). 
		NOTE: See Supplemental File 1 for a protocol on how to activate PBMCs.</li>
		<li>Add the concentrated viral supernatant (~2 &#215; 106 TU, MOI 2) to the cell suspension. The total volume of the cell-virus suspension should not exceed 200 &#181;L for a 48-well scaffold or 350 &#181;L for a 24-well scaffold.</li>
		<li>Add the cell-virus mixture dropwise to the top of the dry scaffold. For negative control experiments, use complete cell culture media in place of the concentrated virus. Add the cell suspension to the top of the dry scaffold.</li>
		<li>Repeat steps 2.2.1-2.2.3 for each scaffold.</li>
		<li>Incubate the scaffolds in a cell culture incubator at 37 &#730;C for 45-60 min. Remove the scaffolds from the incubator; the scaffolds will fully absorb the solution during this time.</li>
		<li>To induce proliferation, add complete cell culture media supplemented with IL-15 (5 ng/mL) and IL-7 (10 ng/mL) to each well; IL-2 can also be used. Add 500 &#181;L to each 48-well scaffold or 1 mL to each 24-well scaffold.</li>
		<li>Incubate the scaffolds at 37 &#730;C for 72 h. As the cells proliferate, the media will turn orange.</li>
	</ol>
	</li>
	<li>Isolate cells from the scaffolds for analysis.
	<ol>
		<li>Dilute 0.5 M EDTA to 0.25 M EDTA in 1x PBS.</li>
		<li>Remove excess medium from each well and collect into separate 15 mL centrifuge tubes.</li>
		<li>To isolate cells from the scaffold, add 0.25 M EDTA to each scaffold. Add 300 &#181;L to 48-well scaffolds or 1 mL to 24-well scaffolds. Let the plate sit or gently agitate for 3-4 min.</li>
		<li>Once the scaffold is mostly dissolved, pipette in and out gently inside the well. A few in-and-out pipetting steps might be necessary to fully dissolve the scaffold. If it does not dissolve in 10 min, add another 200 &#181;L of 0.25 M EDTA.</li>
		<li>Once the scaffold dissolves completely, transfer the solution to a 15 mL centrifuge tube.</li>
		<li>Repeat steps 2.3.4 and 2.3.5 for each scaffold.</li>
		<li>Wash the cells twice by adding 12 mL PBS to each centrifuge tube and centrifuging at 400 &#215; g for 5 min. A cell pellet will form at the bottom of each tube. Aspirate the supernatant and repeat the wash. Be careful to fully aspirate the supernatant with every wash as it is crucial to remove all the EDTA.</li>
		<li>After the second wash with PBS, the cell pellet is now ready for analysis. Follow the appropriate protocol required for the analysis.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Prinzing, B. L., Gottschalk, S. M., Krenciute, G. C. A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+therapy+for+glioblastoma:+ready+for+the+next+round+of+clinical+testing.">T-cell therapy for glioblastoma: ready for the next round of clinical testing.</a> Expert Review of Anticancer Therapy. 18 (5), 451-461 (2018).</li><li>Bagley, S. J., Desai, A. S., Linette, G. P., June, C. H., O'Rourke, D. 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-cell+therapy+for+glioblastoma:+recent+clinical+advances+and+future+challenges.">CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges.</a> Neuro-oncology. 20 (11), 1429-1438 (2018).</li><li>Nair, R., Westin, J. <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.">CAR T cells.</a> Advances in Experimental Medicine and Biology. 1342, 297-317 (2021).</li><li>Jackson, H. J., Rafiq, S., Brentjens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Driving+CAR+T-cells+forward.">Driving CAR T-cells forward.</a> Nature Reviews. Clinical Oncology. 13 (6), 370-383 (2016).</li><li>Sterner, R. C., Sterner, R. 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+cell+therapy:+current+limitations+and+potential+strategies.">CAR-T cell therapy: current limitations and potential strategies.</a> Blood Cancer Journal. 11 (4), 69 (2021).</li><li>Miliotou, A. N., Papadopoulou, L. C. <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+therapy:+A+new+era+in+cancer+immunotherapy.">CAR T-cell therapy: A new era in cancer immunotherapy.</a> Current Pharmaceutical Biotechnology. 19 (1), 5-18 (2018).</li><li>Lee, H. -. 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=Retronectin+enhances+lentivirus-mediated+gene+delivery+into+hematopoietic+progenitor+cells.">Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells.</a> Biologicals: Journal of the International Association of Biological Standardization. 37 (4), 203-209 (2009).</li><li>Rajabzadeh, A., Hamidieh, A. A., Rahbarizadeh, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinoculation+and+retronectin+highly+enhance+the+gene+transduction+efficiency+of+Mucin-1-specific+chimeric+antigen+receptor+(CAR)+in+human+primary+T+cells.">Spinoculation and retronectin highly enhance the gene transduction efficiency of Mucin-1-specific chimeric antigen receptor (CAR) in human primary T cells.</a> BMC Molecular and Cell Biology. 22 (1), 57 (2021).</li><li>Sun, J., Tan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate-based+biomaterials+for+regenerative+medicine+applications.">Alginate-based biomaterials for regenerative medicine applications.</a> Materials. 6 (4), 1285-1309 (2013).</li><li>Nayak, A. K., Mohanta, B. C., Hasnain, M. S., Hoda, M. N., Tripathi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+14+-+Alginate-based+scaffolds+for+drug+delivery+in+tissue+engineering.">Chapter 14 - Alginate-based scaffolds for drug delivery in tissue engineering.</a> Alginates in Drug Delivery. , 359-386 (2020).</li><li>Lee, K. Y., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate:+properties+and+biomedical+applications.">Alginate: properties and biomedical applications.</a> Progress in Polymer Science. 37 (1), 106-126 (2012).</li><li>Kuo, C. K., Ma, P. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionically+crosslinked+alginate+hydrogels+as+scaffolds+for+tissue+engineering:+part+1.+Structure,+gelation+rate+and+mechanical+properties.">Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties.</a> Biomaterials. 22 (6), 511-521 (2001).</li><li>Soccol, 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=Probiotic+nondairy+beverages.">Probiotic nondairy beverages.</a> Handbook of Plant-Based Fermented Food and Beverage Technology, Second Edition. , 707-728 (2012).</li><li>Moody, C. 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=Restoring+carboxylates+on+highly+modified+alginates+improves+gelation,+tissue+retention+and+systemic+capture.">Restoring carboxylates on highly modified alginates improves gelation, tissue retention and systemic capture.</a> Acta Biomaterialia. 138, 208-217 (2022).</li><li>Brudno, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replenishable+drug+depot+to+combat+post-resection+cancer+recurrence.">Replenishable drug depot to combat post-resection cancer recurrence.</a> Biomaterials. 178, 373-382 (2018).</li><li>Moody, C. T., Palvai, S., Brudno, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+cross-linking+improves+retention+and+targeting+of+refillable+alginate+depots.">Click cross-linking improves retention and targeting of refillable alginate depots.</a> Acta Biomaterialia. 112, 112-121 (2020).</li><li>Agarwalla, P., 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=Scaffold-mediated+static+transduction+of+T+Cells+for+CAR-T+Cell+therapy.">Scaffold-mediated static transduction of T Cells for CAR-T Cell therapy.</a> Advanced Healthcare Materials. 9 (14), 2000275 (2020).</li><li>Agarwalla, P., 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=Bioinstructive+implantable+scaffolds+for+rapid+in+vivo+manufacture+and+release+of+CAR-T+cells.">Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells.</a> Nature Biotechnology. 40 (8), 1250-1258 (2022).</li><li>Vera, 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=T+lymphocytes+redirected+against+the+kappa+light+chain+of+human+immunoglobulin+efficiently+kill+mature+B+lymphocyte-derived+malignant+cells.">T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells.</a> Blood. 108 (12), 3890-3897 (2006).</li><li>. PRONOVA UP MVG. IFF Nutrition Norge AS Available from: <a target="_blank" href="https://novamatrix.biz/store/pronova-up-mvg/">https://novamatrix.biz/store/pronova-up-mvg/</a> (2022)</li><li>Zappasodi, R., Budhu, S., Abu-Akeel, M., Merghoub, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assays+for+effector+T+cell+functions+and+activity+of+immunomodulatory+antibodies.">In vitro assays for effector T cell functions and activity of immunomodulatory antibodies.</a> Methods in Enzymology. 631, 43-59 (2020).</li><li>Kong, B. S., Lee, C., Cho, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+assessment+of+human+T+cell+activation+by+real-time+metabolic+flux+analysis.">Protocol for the assessment of human T cell activation by real-time metabolic flux analysis.</a> STAR Protocols. 3 (1), 101084 (2022).</li><li>Bio-Rad cell activation protocols. Bio-Rad Available from: <a target="_blank" href="https://www.bio-rad-antibodies.com/cell-activation.html?JSESSIONID_STERLING=D6E538F76818E53C29884D6CC7334F24">https://www.bio-rad-antibodies.com/cell-activation.html?JSESSIONID_STERLING=D6E538F76818E53C29884D6CC7334F24</a> (2022)</li><li>Lin, H. -. R., Yeh, Y. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+alginate/hydroxyapatite+composite+scaffolds+for+bone+tissue+engineering:+Preparation,+characterization,+andin+vitro+studies.">Porous alginate/hydroxyapatite composite scaffolds for bone tissue engineering: Preparation, characterization, andin vitro studies.</a> Journal of Biomedical Materials Research. 71 (1), 52-65 (2004).</li><li>Wu, J., Zhao, Q., Sun, J., Zhou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+poly(ethylene+glycol)+aligned+porous+cryogels+using+a+unidirectional+freezing+technique.">Preparation of poly(ethylene glycol) aligned porous cryogels using a unidirectional freezing technique.</a> Soft Matter. 8 (13), 3620 (2012).</li></ol>

P.A. and Y.B. are inventors on patents related to the use of biomaterials for generation of CAR-T cell therapeutics. Y.B. receives an industry-sponsored research grant related to CAR-T cell therapeutic technology (unrelated to this work).&#160;All other authors declare that they have no competing interests.

CAR-T cell therapy continues to gain interest for both research and commercial applications. Despite the success CAR-T cell therapy has had in treating blood cancers, the high cost of the procedure limits its use. The protocol presented here introduces a new method for viral gene transfer of T cells without the need for spinoculation of retronectin-coated plates. Producing dry macroporous alginate scaffolds to mediate transduction is relatively simple and is a suitable low-cost replacement for the conventional method.</p...

Immunotherapy has emerged as a revolutionary cancer treatment paradigm due to its ability to specifically target tumors, limit off-target cytotoxicity, and prevent relapse. Particularly, chimeric antigen receptor T (CAR-T) cell therapy has gained popularity due to its success in treating lymphomas and leukemias. The FDA approved the first CAR-T cell therapy in 2017, and, since then, has approved four more CAR-T cell therapies1,2,3,4,5. CARs have an antigen recognition domain usually consisting of a single chain variable fragment of a monoclonal antibody that is specific for a tumor associated antigen3,4. When a CAR interacts with its tumor-associated antigen, the CAR-T cells become activated, leading to an antitumor response involving cytokine release, cytolytic degranulation, transcription factor expression, and T cell proliferation. To produce CAR-T cells, blood is collected from the patient to obtain their T cells. CARs are genetically added to the patient&#39;s T cells using a virus. The CAR-T cells are grown in vitro and infused back into the patient2,3,4,6. Successful generation of CAR-T cells is determined by the transduction efficiency, which describes the number of T cells that are genetically modified into CAR-T cells.
Currently, the gold standard for CAR-T cell generation is spinoculation of activated T cells and virus on retronectin-coated plates7,8. Transduction begins when viral particles engage with the surface of the T cells. Retronectin promotes colocalization of virus and cells by increasing the binding efficiency between the viral particles and the cells, enhancing transduction7,8. Retronectin does not work well on its own and needs to be accompanied by spinoculation, which enhances gene transfer by concentrating the viral particles and increasing the surface permeability of the T cell, allowing for easier viral infection8. Despite the success of spinoculation on retronectin-coated plates, it is a complex process that requires multiple spin cycles and expensive reagents. Therefore, alternate methods for viral gene transfer that are quicker and cheaper are highly desirable.
Alginate is a natural anionic polysaccharide extensively used in the biomedical industry due to its low cost, good safety profile, and ability to form hydrogels upon mixing with divalent cations9,10,11,12. Alginate is a GMP-compliant polymer and is generally recognized as safe (GRAS) by the FDA13. Cross-linking alginate with cations creates stable hydrogels often used in wound healing, delivery of small chemical drugs and proteins, and cell transportation9,10,11,12,14,15,16. Due to its excellent gelling properties, alginate is the preferred material to create porous scaffolds by freeze-drying10,17. These characteristics of alginate make it an attractive candidate for producing a scaffold that can mediate viral gene transfer of activated cells.
Described here is a protocol for making dry macroporous alginate scaffolds, known as Drydux scaffolds, that statically transduce T cells by viral gene transfer17,18. The process for making these scaffolds is shown in Figure 1. These scaffolds eliminate the need for spinoculation of retronectin-coated plates. The macroporous alginate scaffolds encourage the interaction of viral particles and T cells to enable efficient gene transfer in a single step without affecting functionality and viability of the engineered T cells17. When followed correctly, these macroporous alginate scaffolds have a transduction efficiency of at least 80%, simplifying and shortening the viral transduction process.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64036/64036fig01v2.jpg" /> 
Figure 1: Schematic and timeline of the protocol. (A) Timeline for making the dry macroporous alginate scaffolds. Alginate is cross-linked with calcium-D-gluconate and frozen overnight. The frozen scaffolds are lyophilized for 72 h to create the Drydux scaffolds. (B) Timeline for viral transduction of activated cells. Activated cells and virus (MOI 2) are seeded on top of the scaffold and incubated in complete media supplemented with IL-7 and IL-15. The scaffolds absorb the mixture and promote viral gene transfer. EDTA is used to dissolve the scaffolds and isolate the transduced cells. After washing twice with PBS, the cell pellet can be used for analysis. Abbreviations: PBS = phosphate-buffered saline; PBMCs = peripheral blood mononuclear cells. <a href="https://www.jove.com/files/ftp_upload/64036/64036fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.5 M EDTA</td><td>Invitrogen</td><td>15575-038</td><td>UltraPure, pH 8.0</td></tr><tr><td>1x DPBS</td><td>Gibco</td><td>14190-144</td><td>No calcium chloride or magnesium chloride</td></tr><tr><td>3% Acetic Acid with Methylene Blue</td><td>Stemcell Technologies Inc</td><td>07060</td><td></td></tr><tr><td>Activated Periphreal Blood Mononuclear Cells</td><td>-</td><td>-</td><td>Fresh or frozen</td></tr><tr><td>Calcium-D-Gluconate</td><td>Alfa Aesar</td><td>A11649</td><td></td></tr><tr><td>CD28.2 Antibody</td><td>BD</td><td>555725</td><td>1 mg/mL</td></tr><tr><td>CD3 Antibody</td><td>Miltenyi</td><td>130-093-387</td><td>100 &amp;mu;g/mL</td></tr><tr><td>Click&#039;s Media</td><td>FUJIFILM IRVINE SCIENTIFIC MS</td><td>9195</td><td></td></tr><tr><td>DI Water</td><td>-</td><td>-</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35-050-061</td><td></td></tr><tr><td>HyClone FBS</td><td>Cytvia</td><td>SH3039603</td><td></td></tr><tr><td>HyClone RPMI 1640 Media</td><td>Cytvia</td><td>SH3009601</td><td></td></tr><tr><td>Penicillin-streptomycin (P/S)</td><td>Gibco</td><td>15-140-122</td><td></td></tr><tr><td>Peripheral Blood Mononuclear Cells</td><td>-</td><td>-</td><td>Fresh or frozen</td></tr><tr><td>PRONOVA UP MVG</td><td>NovaMatrix</td><td>4200101</td><td>Sodium alginate</td></tr><tr><td>Recombinant Human IL-15</td><td>Peprotech</td><td>200-15</td><td>5 ng/mL</td></tr><tr><td>Recombinant Human IL-7</td><td>Peprotech</td><td>200-07</td><td>10 ng/mL</td></tr><tr><td>Retrovirus</td><td>-</td><td>-</td><td>1 x 10&lt;sup&gt;6&lt;/sup&gt; TU/mL</td></tr></tbody></table>

fabrication and use of dry macroporous alginate scaffolds for viral transduction of t cells

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce &gt;85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.

These macroporous alginate scaffolds are easy to make and should come out of the lyophilizer as porous, fluffy, and white discs. Although not studied in this experiment, calcium-alginate solution can be cast into different molds to create scaffolds of varying shapes, depending on the needs of the user9,10. The scaffolds are electrostatic and may stick to the lid of the well-plate or to a gloved finger. Figure 2 demonstrates what the ...

Watch this Scientific Journal Video about Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells at JoVE.com

Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells

Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with &gt;85% transduction.

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by ...

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2.7K Views.  University of North Carolina at Chapel Hill and North Carolina State University. Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with >85% transduction.

Video: Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

Environment

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Immunology and Infection

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...