Name: fabrication and use of dry macroporous alginate scaffolds for viral transduction of t cells
Uploaded: 2022-09-09T13:00:00
Description: Watch this Scientific Journal Video about Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells at JoVE.com

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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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 ...

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