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

这项工作得到了美国国立卫生研究院通过拨款编号R37-CA260223，R21CA246414的支持。我们感谢NCSU流式细胞术核心对流式细胞术分析的培训和指导。原理图是用 Biorender.com 创建的

所有涉及人类原代细胞和逆转录病毒载体的程序均符合北卡罗来纳州立大学的生物安全指南，并得到环境健康与安全办公室的批准。人外周血单核细胞是从商业来源购买的血沉棕黄层。原代人类细胞必须从人血沉棕黄层部分中分离出来，并需要生物安全2级许可和详细的标准操作程序以及工作所在机构的批准。病毒载体，包括用于如前所述制备的转导的逆转录病毒载体上清液19，?...

<ol>
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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. 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CAR-T细胞疗法继续引起人们对研究和商业应用的兴趣。尽管CAR-T细胞疗法在治疗血癌方面取得了成功，但该程序的高成本限制了其使用。这里介绍的方案介绍了一种用于T细胞病毒基因转移的新方法，而无需对retronectin包被的板进行棘刺。生产干燥的大孔海藻酸盐支架来介导转导相对简单，是传统方法的合适低成本替代品。
有时，支架外观可能与图2所示不...

免疫疗法已成为一种革命性的癌症治疗范例，因为它能够特异性靶向肿瘤、限制脱靶细胞毒性和预防复发。特别是嵌合抗原受体T（CAR-T）细胞疗法因其在治疗淋巴瘤和白血病方面的成功而广受欢迎。FDA于2017年批准了第一种CAR-T细胞疗法，此后又批准了四种CAR-T细胞疗法1，2，3，4，5。CAR 具有抗原识别结构域，通常由单克隆抗体的单链可变片段组成，该片段对肿瘤相关抗原3，4 具有特异性。当CAR与其肿瘤相关抗原相互作用时，CAR-T细胞被激活，导致涉及细胞因子释放，细胞溶解脱颗粒，转录因子表达和T细胞增殖的抗肿瘤反应。为了产生CAR-T细胞，从患者身上收集血液以获得他们的T细胞。CAR是使用病毒遗传添加到患者的T细胞中的。CAR-T细胞在体外生长并输回患者体内2，3，4，6。CAR-T细胞的成功生成取决于转导效率，转导效率描述了转基因成CAR-T细胞的T细胞数量。
目前，CAR-T细胞生成的金标准是活化的T细胞和病毒在再连蛋白包被的平板7，8上的棘化。当病毒颗粒与T细胞表面接触时，转导开始。Retronectin通过增加病毒颗粒和细胞之间的结合效率来促进病毒和细胞的共定位，增强转导7，8。Retronectin本身不能很好地工作，需要伴随着脊髓，这通过浓缩病毒颗粒和增加T细胞的表面通透性来增强基因转移，从而使病毒感染更容易8。尽管在 retronectin 包被的板上成功了脊髓成形，但这是一个复杂的过程，需要多次旋转循环和昂贵的试剂。因此，非常需要更快、更便宜的病毒基因转移替代方法。
海藻酸盐是一种天然阴离子多糖，由于其成本低、安全性好以及与二价阳离子9，10，11，12 混合后形成水凝胶的能力，广泛用于生物医学行业。海藻酸盐是一种符合 GMP 标准的聚合物，通常被 FDA13 认可为安全 （GRAS）。海藻酸盐与阳离子交联产生稳定的水凝胶，通常用于伤口愈合，小化学药物和蛋白质的递送以及细胞运输9，10，11，12，14，15，16。由于其优异的胶凝性能，海藻酸盐是通过冷冻干燥制造多孔支架的首选材料10，17。藻酸盐的这些特性使其成为生产可以介导活化细胞病毒基因转移的支架的有吸引力的候选者。
这里描述的是用于制造干大孔藻酸盐支架的方案，称为 Drydux 支架，其通过病毒基因转移静态转导 T 细胞17，18。制作这些支架的过程如图 1所示。这些支架消除了对再连接蛋白涂层板的棘刺形成的需要。大孔藻酸盐支架促进病毒颗粒和T细胞的相互作用，从而在一步中实现高效的基因转移，而不会影响工程T细胞的功能和活力17。如果正确遵循，这些大孔藻酸盐支架的转导效率至少为80%，简化和缩短了病毒转导过程。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64036/64036fig01v2.jpg"> 图 1：协议的示意图和 时间表。 （A）制作干式大孔海藻酸盐支架的时间表。海藻酸盐与钙-D-葡萄糖酸交联并冷冻过夜。将冷冻支架冻干72小时以创建Drydux支架。（B）活化细胞病毒转导的时间表。将活化的细胞和病毒（MOI 2）接种在支架顶部，并在补充有IL-7和IL-15的完整培养基中孵育。支架吸收混合物并促进病毒基因转移。EDTA用于溶解支架并分离转导细胞。用PBS洗涤两次后，细胞沉淀可用于分析。缩写：PBS = 磷酸盐缓冲盐水;PBMC=外周血单核细胞。 <a href="https://www.jove.com/files/ftp_upload/64036/64036fig01largev2.jpg" target="_blank">请点击此处查看此图的大图。</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

在过去几年中，用于CAR-T细胞治疗的T细胞基因工程已成为癌症治疗的最前沿。CAR-T细胞是通过病毒基因转移到T细胞中产生的。目前病毒基因转移的黄金标准涉及再连蛋白包被板的棘刺，这既昂贵又耗时。非常需要高效且具有成本效益的方法来生成CAR-T细胞。这里描述的是一种制造廉价、干燥的大孔海藻酸盐支架的方法，称为 Drydux 支架，可有效促进活化 T 细胞的病毒转导。支架设计用于代替接种病毒的再连蛋白包被板的金标准棘刺，并简化转导细胞的过程。海藻酸盐与钙-D-葡萄糖酸盐交联并冷冻过夜以产生支架。将冷冻支架在冻干机中冷冻干燥72小时，以完成干燥的大孔支架的形成。当病毒和活化的T细胞一起接种在支架顶部以产生转基因细胞时，支架介导病毒基因转移。支架产生>85%的原代T细胞转导，这与棘皮膜在Retronectin包被的板上的转导效率相当。这些结果表明，干式大孔海藻酸盐支架是传统转导方法的更便宜、更方便的替代方案。

这些大孔藻酸盐支架易于制作，应以多孔、蓬松和白色圆盘的形式从冻干机中出来。虽然没有在这个实验中研究，但海藻酸钙溶液可以铸造到不同的模具中，以创建不同形状的支架，具体取决于用户的需要9，10。支架是静电的，可能会粘在孔板的盖子上或戴手套的手指上。 图 2 演示了脚手架完成后的外观。图中还显示了 24 孔和 48...

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

本文是用于创建干燥的大孔藻酸盐支架的方案，该支架介导有效的病毒基因转移，用于T细胞的基因工程，包括用于CAR-T细胞治疗的T细胞。支架被证明以>85%的转导转导活化的原代T细胞。

干式大孔海藻酸盐支架的制备和使用用于T细胞的病毒转导

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 ...

本视频中描述的是一种用于创建和使用称为 Drydux 的干大孔藻酸盐支架的方案，可有效促进活化 T 细胞的转导。Drydux 旨在代替接种病毒的逆转录凝连蛋白包被板的金标准棘刺，并简化细胞转导过程。Drydux 的转导效率可与 RetroNectin 包被板上的脊髓相媲美，并且是传统转导方法更便宜、更方便的替代方案。首先，制备0.4%的葡萄糖酸钙溶液。将无菌过滤去离子水加入烧杯中的D-葡萄糖酸钙中。中速搅拌直至葡萄糖酸钙溶解，大约需要 1 1/2 小时。溶解后，无菌过滤钙-D-葡萄糖酸溶液。该溶液可以在室温下储存。接下来，准备2%海藻酸盐溶液。这可以使用螺旋盖小瓶或烧杯来完成。两者都将在此处显示。小心地将超纯海藻酸盐添加到容器中。在海藻酸盐中加入适量的去离子水。选择不会妨碍混合的最大搅拌棒并将其添加到容器中。将溶液搅拌到海藻酸盐溶解，这大约需要一个小时。如果容器侧面有任何大的藻酸盐团块，请将容器侧向倾斜以将其移开。小团块会在一小时内溶解，无需担心。一旦2%海藻酸盐溶液溶解，降低搅拌速度。将等体积的钙-D-葡萄糖酸盐溶液缓慢添加到海藻酸盐溶液中。缓慢添加将有助于防止形成交联团块。加入葡萄糖酸钙溶液后，加快搅拌速度，剧烈搅拌15分钟。将容器侧向倾斜以去除可能已形成的任何团块。剧烈搅拌15分钟后，确保溶液中没有团块。将溶液移液到48孔板或24孔板中。对于 48 孔板，将 300 微升移液到每个孔中。确保缓慢施洗并经常更换吸头，因为溶液会很粘稠，并且可能会粘在吸头上。对于 24 孔板，将一毫升移入每个孔中。同样，缓慢施放，并经常更换移液器吸头。盖上孔板，在负20摄氏度下冷冻过夜。通过取下盖子并用湿巾和橡皮筋固定顶部来准备冻干机的板。将板放入冻干管中，然后放在冻干机上72小时。72小时后，从冻干机中取出板。支架现在已准备好用于转导。如果不立即使用脚手架，请用石蜡膜密封板并储存在四摄氏度下，直到需要为止。对于长期储存，除了用封口膜密封外，建议对板进行真空密封。通过将两毫升病毒溶液通过离心过滤器以1， 500g离心10分钟来浓缩病毒上清液。每个支架需要 100 到 200 微升浓缩病毒。如果浓缩的病毒溶液体积大于 200 微升，再旋转几分钟。对于每个支架，将 100 万个活化的 T 细胞悬浮在 50 微升的完整细胞培养基中等分试样。将浓缩的病毒添加到细胞悬液中。对于48孔支架，细胞病毒悬浮液的总体积不应超过200微升，对于24孔支架，细胞病毒悬浮液的总体积不应超过350微升。将细胞病毒混合物滴加到干支架的顶部。对于阴性对照实验，使用完整的细胞培养基代替浓缩的病毒。将此细胞悬液添加到干支架的顶部。将支架在 37 摄氏度的细胞培养箱中孵育 45 至 60 分钟。从培养箱中取出支架。在此期间，支架应完全吸收溶液。为了诱导增殖，向每个孔中加入补充有IL-7和IL-15的完整细胞培养基。也可以使用IL-2。对于 24 孔支架，添加一毫升。对于 48 孔支架，添加 500 微升。在 37 摄氏度下孵育 72 小时。细胞将在72小时内增殖，这可能会使培养基变成橙色。从每个孔中取出多余的培养基。要从支架中分离细胞，请向每个支架添加0.25摩尔EDTA溶液。24孔支架加1毫升，48孔支架加300微升。让盘子静置，或轻轻搅拌三到四分钟。一旦大部分溶解，在孔内轻轻移入和移出。可能需要一些进出移液步骤才能完全溶解支架。将溶液转移到15毫升离心管中。用PBS清洗细胞两次。向每个离心管中加入12毫升PBS。以400克离心五分钟。细胞沉淀将在管底部形成。吸出上清液，并重复洗涤。每次洗涤时都要小心完全去除上清液，因为去除所有EDTA至关重要。用PBS第二次洗涤后，细胞沉淀即可进行分析。采用流式细胞术测定各组转导效率。非转导细胞用作阴性对照，未显示转导。接种在没有GFP逆转录病毒的支架上的细胞也没有显示转导。Drydux组将活化的细胞接种到干燥的大孔藻酸盐支架上，显示出85%的转导，略低于RetroNectin组。这些结果表明，Drydux支架通过逆转录病毒基因转移有效地转导细胞，而无需逆转录菌素包被的平板进行棘刺。Drydux支架表现出与逆转录菌素相当的高转导效率，为转导T细胞提供了一种替代方法。由于制造Drydux支架的成本低，该过程有可能显着降低CAR-T细胞疗法的成本。

Research

JoVE Journal

Bioengineering

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

科研

生物工程

Use of Human Perivascular Stem Cells for Bone Regeneration

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

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

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

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

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Melt Electrospinning Writing of Three-dimensional PolyÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Âµ-caprolactone Scaffolds with Controllable Morphologies for Tissue Engineering Applications

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

在保持中央记忆表型的九天内，原T细胞的转导与扩展

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

来自微凝胶棒的互连大孔3D支架

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