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 w...

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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. 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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Click&#39;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 106 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 ...

Described in this video is a protocol for creating and using dry macroporous alginate scaffolds known as Drydux that efficiently promote transduction of activated T-cells. Drydux is designed to be used in place of gold standard spinoculation of RetroNectin-coated plates seeded with virus and simplifies the process for transducing cells. Drydux has a transduction efficiency comparable to spinoculation on RetroNectin-coated plates, and serves as a cheaper and more convenient alternative to the conventional transduction method.First, prepare a 0.4%solution of calcium-D-gluconate. Add sterile filtered DI water to calcium-D-gluconate in a beaker. Stir on medium speed until the calcium-D-gluconate has dissolved, which takes approximately 1 1/2 hours.Once dissolved, sterile filter the calcium-D-gluconate solution. This solution can be stored at room temperature. Next, prepare a 2%alginate solution.This can be accomplished using a screw cap vial, or a beaker. Both will be shown here. Carefully add the ultrapure alginate to the vessel.Add the appropriate amount of DI water to the alginate. Choose the largest stir bar possible that will not impede mixing and add it to the vessel. Stir the solution on high until the alginate dissolves, which takes approximately one hour.If there are any large alginate clumps on the side of the vessel, tilt the vessel sideways to dislodge them. Small clumps will dissolve over the hour, and are not cause for concern. Once the 2%alginate solution dissolves, reduce the stirring speed.Slowly add an equal volume of the calcium-D-gluconate solution to the alginate solution. Adding it slowly will help prevent cross-linking clumps from forming. When the calcium-gluconate solution has been added, increase the stirring speed, and stir vigorously for 15 minutes.Tilt the vessel sideways to remove any clumps that may have formed. After stirring vigorously for 15 minutes, ensure there are no clumps in the solution. Pipette the solution into a 48, or 24-well plate.For a 48-well plate, pipette 300 microliters into each well. Ensure to cast slowly and change tips often, as the solution will be viscous, and may stick to the tip. For a 24-well plate, pipette one milliliter into each well.Again, cast slowly, and change pipette tips often. Cover the well plates with a lid, and freeze at negative 20 degrees Celsius overnight. Prepare the plates for the lyophilizer by removing the lids and securing the top with wipes and rubber bands.Place the plates in the lyophilizer tube, and put on the lyophilizer for 72 hours. After 72 hours, remove the plate from the lyophilizer. The scaffolds are now ready to be used for transduction.If not using the scaffolds immediately, seal the plate with Parafilm and store at four degrees Celsius until needed. For long-term storage, it is recommended to vacuum seal the plate in addition to sealing with Parafilm. Concentrate viral supernatant by centrifuging two milliliters of viral solution through a centrifugal filter at 1, 500 g for 10 minutes.100 to 200 microliters of concentrated virus is needed per scaffold. Spin for a few additional minutes if the concentrated virus solution volume is greater than 200 microliters. For each scaffold, make an aliquot of 1 million activated T-cells suspended in 50 microliters of complete cell culture media.Add the concentrated virus to the cell suspension. The total volume of the cell virus suspension should not exceed 200 microliters for a 48-well scaffold, or 350 microliters for a 24-well scaffolded. Add the cell virus mixture drop-wise to the top of the dry scaffold.For negative control experiments, use complete cell culture media in place of concentrated virus. Add this cell suspension to the top of the dry scaffold. Incubate the scaffolds in the cell culture incubator at 37 degrees Celsius for 45 to 60 minutes.Remove the scaffolds from the incubator. The scaffolds should fully absorb the solution during this time. To induce proliferation, add complete cell culture media supplemented with IL-7 and IL-15 to each well.IL-2 can also be used. For a 24-well scaffold, add one milliliter. For a 48-well scaffold, add 500 microliters.Incubate at 37 degrees Celsius for 72 hours. The cells will proliferate over the 72 hours, which may turn the media orange. Remove excess media from each well.To isolate cells from the scaffold, add 0.25 molar EDTA solution to each scaffold. Add one milliliter for 24-well scaffolds, or 300 microliters for 48-well scaffolds. Let the plate sit, or gently agitate for three to four minutes.Once 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. Transfer the solution to a 15-milliliter centrifuge tube.Wash the cells twice with PBS. Add 12 milliliters of PBS to each centrifuge tube. Centrifuge at 400 g for five minutes.A cell pellet will form at the bottom of the tube. Aspirate the supernatant solution, and repeat the wash. Be careful to fully remove supernatant with every wash as it is crucial to get rid of all EDTA.After washing with PBS a second time, the cell pellet is ready for analysis. Flow cytometry was used to determine the transduction efficiency of each group. Non-transduced cells were used as a negative control, and showed no transduction.Cells seeded on scaffolds without GFP retrovirus also showed no transduction. The Drydux group, which had activated cells seeded onto the dry macroporous alginate scaffolds, showed 85%transduction, just lower than the RetroNectin group. These results demonstrate that Drydux scaffolds effectively transduced cells by retroviral gene transfer without the need for spinoculation of RetroNectin-coated plates.Drydux scaffolds exhibit high transduction efficiency comparable to RetroNectin, providing an alternative method for transducing T-cells. Due to the low cost of manufacturing the Drydux scaffolds, this process has the potential to significantly lower the cost of CAR T-cell therapies.

fabrication-use-dry-macroporous-alginate-scaffolds-for-viral

Research

JoVE Journal

Bioengineering

2.5K visualizações.  University of North Carolina at Chapel Hill and North Carolina State University. Descrito neste vídeo é um protocolo para a criação e uso de andaimes de alginato macroporoso seco conhecidos como Drydux que promovem eficientemente a transdução de células T ativadas. O Drydux foi projetado para ser usado no lugar da espinoculação padrão-ouro de placas revestidas com RetroNectin, semeadas com vírus e simplifica o processo de transdução de células. O Drydux tem uma eficiência de transdução comparável à espinoculação em placas revestidas com RetroNectin, e...