我们感谢 L. Brockmann 对手稿的严格审阅。这项工作得到了 NIH 1R01EB030352 和 UL1 TR001873 的支持。

所有动物程序均在机构动物护理和使用委员会（哥伦比亚大学，协议AC-AABQ5551和AC-AAAZ4470）的批准下使用体重在20-25克之间的6-8周龄雌性BALB / c或CF57BL / 6小鼠进行。这些动物是从商业来源获得的（见 材料表）。该协议围绕小鼠 T 细胞的“激活后天数”构建，病毒产生从第 -2 天开始。逆转录病毒在初始生产后可以储存在-80°C，并且对于该方案的未来使用，可以从步骤2开始，在第0天分离和激活T细胞。
1. 逆转录病毒载体生产
注意：通过将包装基因分离成两个单独的质粒，使病毒产物具有复制缺陷（参见 材料表），大大降低了重组事件和无意中产生具有复制能力的病毒的可能性。
<ol><li>在转染前一天（第 -2 天程序）准备 Phoenix Eco 细胞。<ol><li>使用 30 mL 培养基（Phoenix Eco 培养基，详见表 1）将大约 1 x 107 个细胞放入 15 cm TC 处理的平板或 T150 培养瓶中。</li>
		<li>在37°C孵育过夜。 18-24小时后，细胞应约70%汇合并均匀分布，以确保高病毒产量而不会过度生长。 注意：为获得最佳结果，请使用传代率低的细胞，并在接种前一天传代用于病毒生产。在常规培养过程中，不要让Phoenix Eco细胞过度生长。</li>
	</ol></li>
	<li>在还原血清培养基中制备含有脂质转染和增强子试剂、pCL-Eco （Gag/Pol） 和 pMSCV 表达质粒 （pMSCV_PGK_mGFP28z） 的转染混合物（参见 材料表）（第 -1 天程序）。 注意：共转染应以 pMSCV 和 pCL-Eco 的 1：1 比例进行。<ol><li>准备试管 A：每 15 cm 板将 105 μL 转染试剂稀释在 3.75 mL 还原血清培养基中，并通过涡旋或上下移液充分混合。</li>
		<li>准备管B：用90μL增强剂试剂将pMSCV表达质粒（21μg）和pCL-Eco（21μg）稀释到每15cm板3.75mL还原血清培养基中。通过上下移液充分混合。 注意：如果产生多种病毒产物，请设置含有 pCL-Eco 和增强剂试剂的预混液。</li>
		<li>将试管 A 加入试管 B 中，用移液管充分混合。在室温下孵育 10-20 分钟，总体积约为 7.5 mL。</li>
		<li>小心地从 Phoenix Eco 培养板中取出 10 mL 细胞培养基，并通过倾斜板和滴液将整个体积的转染混合物添加到剩余的培养基中。将细胞送回37°C培养箱。</li>
	</ol></li>
	<li>在转染后16-20小时（第0天程序）更换转染的Phoenix Eco细胞上的培养基。<ol><li>使用水或珠浴将不含β巯基乙醇的血清和-巯基乙醇（2-ME，参见 材料表）的鼠T细胞培养基（mTCM-病毒收获，如 表1所示）预热至37°C。</li>
		<li>倾斜平板并将移液器吸头放在底角，小心地取出Phoenix Eco培养基，如果可能的话，使用真空吸尘器。避免细胞过度干燥。</li>
		<li>通过在最慢的设置下移液 30 mL，将加热的 mTCM 病毒收获培养基轻轻加入倾斜板的侧面。将细胞送回37°C培养箱。 注意：此步骤会导致Phoenix Eco细胞从平板上脱落并降低病毒滴度。预热的培养基和轻柔的移液将最大限度地减少干扰。</li>
	</ol></li>
	<li>转染后48小时收获病毒上清液（第1天程序）。<ol><li>收获病毒上清液并通过0.45μmPVDF过滤器过滤（参见 材料表）以去除细胞和碎片。分装并在-80°C下冷冻病毒，如果使用新鲜病毒，则直接进行到步骤3的第1天。</li>
		<li>如前所述，通过流式细胞术测定病毒滴度（转导单位/mL）9。此步骤是可选的。<ol><li>通过在非组织培养 （TC） 处理的 96 孔板中小规模转导 1 x 105 个活化的小鼠 T 细胞，预涂有逆转录肾素（参见 材料表）并在最终体积为 100 μL 的 mTCM 病毒收获培养基中加载三倍连续稀释的逆转录病毒上清液来测定病毒滴度。 注意：有关 T 细胞分离、活化和转导的详细说明，请参阅下面的步骤 2 和步骤 3。</li>
			<li>通过流式细胞术在转导后 4-5 天测定 CAR 表面表达。</li>
			<li>根据公式10计算病毒滴度：（N x F x D）/V，其中N是转导的细胞数，F是CAR阳性细胞的频率，D是稀释因子，V是以mL为单位的转导体积，得到转导单位（TU）/mL。 注意：冻融时病毒滴度可能会降低;因此，理想情况下，病毒滴度是在冷冻并随后解冻的病毒上测定的。</li>
		</ol></li>
	</ol></li>
</ol>2. 小鼠T细胞分离
<ol><li>分离并激活小鼠 T 细胞（第 0 天程序）。 注意：这些步骤可以在室温（RT）或4°C下进行，并且应在无菌环境中进行。<ol><li>如前所述，从感兴趣的小鼠品系（例如，Balb / c，C57BL / 6）中收获脾脏11 ，并通过机械解离获得单细胞悬浮液。</li>
		<li>使用无菌注射器的背面，通过 70-100 μm 细胞过滤器将脾脏粉碎到 50 mL 锥形管中。</li>
		<li>将注射器放在一边后，用 5 mL T 细胞分离缓冲液（磷酸盐缓冲盐水，PBS，补充有 2% 胎牛血清，FBS）洗涤过滤器。</li>
		<li>重复糖化步骤，然后用 5 mL T 细胞分离缓冲液再次洗涤过滤器。用 T 细胞分离缓冲液将最终体积降至 50 mL。</li>
		<li>使用手动血细胞计数器或自动细胞计数器以 1：20 的比例稀释，然后以 1：1 的比例与台盼蓝混合，对活细胞进行计数。</li>
		<li>通过在4°C（或RT）下以450× g离心10分钟来沉淀细胞，如果使用推荐的T细胞分离试剂盒，则以1×108/ mL重悬脾细胞（参见 材料表）。 注意：在这个阶段，可以通过40-70μm过滤器重新过滤脾细胞以去除团块。</li>
	</ol></li>
	<li>按照制造商的说明通过阴性选择分离CD3 + T细胞（参见 材料表）。磁分离后，将分离物转移到 15 mL 锥形管中并进行最终活细胞计数。</li>
	<li>通过在4°C（或RT）下以450× g离心10分钟的方式沉淀分离的T细胞，并将其以1×106/ mL重悬于mTCM活化培养基中（表1）。</li>
	<li>通过以 25 μL/1 x 106 个 T 细胞和 100 U/mL IL-2 的比例加入小鼠抗 CD3/抗 CD28 单克隆抗体包被磁珠（参见材料表）来激活 T 细胞。</li>
	<li>将细胞置于37°C的培养箱中，并放置过夜。 注意：由于 T 细胞的体积小，可以通过以 1：10-1：20 的比例稀释并以 1：1 的比例与台盼蓝混合以使用血细胞计数器进行手动计数来实现更准确的活 T 细胞计数。纯度可以通过流式细胞术通过用荧光偶联的 αCD3 抗体对细胞进行染色来确定。每个脾脏将产生 7-10 x 106 个 T 细胞，具体取决于小鼠的年龄和品系。</li>
</ol>3. 小鼠T细胞转导
<ol><li>准备用于转导的板（第 0 天程序）。<ol><li>用0.5mL人纤连蛋白转导增强剂试剂（参见 材料表）预涂未经处理的无菌24孔板，终浓度为20-40μg/ mL，将其稀释在无菌PBS中，并在4°C下储存过夜。 注意：该步骤也可以在第 1 天通过用转导试剂包被未处理的 24 孔板并在室温下孵育 2 小时来执行。</li>
	</ol></li>
	<li>进行 T 细胞转导（第 1 天程序）。<ol><li>准备用于转导的预涂层板。从预包被的24孔板的每个孔中取出转导试剂，并在室温下用等体积的无菌过滤PBS + 2%牛血清白蛋白（BSA）（0.5mL）封闭30分钟。</li>
		<li>用 0.5-1 mL PBS 洗涤一次。</li>
	</ol></li>
	<li>将来自步骤1的0.5-1mL纯逆转录病毒或根据病毒滴度稀释到每个预包被孔中，并在2,000× g 和32°C下离心90分钟。</li>
	<li>向每个病毒加载的孔中加入1mL活化的T细胞，并在450× g 和32°C下离心10分钟。 将细胞送回37°C培养箱过夜。</li>
	<li>转导后24小时，除去1-1.5mL细胞培养基，并用1-1.5mL mTCM完全和10ng / mL重组人IL-7和IL-15代替（见 材料表）。将细胞返回至37°C培养箱（第2天程序）。 注意：在 离体 培养期间，必须每 48 小时向培养物中加入 10 ng/mL 细胞因子，并且 T 细胞不应稀释超过 1 x 106/mL。</li>
	<li>转导后48小时，将细胞从转导板转移到新鲜的24孔或6孔板中，并将细胞返回到37°C培养箱（第3天程序）。 注意：T细胞活力可以通过在转导后24小时至2天转移细胞来改善，具体取决于起始T细胞活力和病毒滴度。</li>
	<li>去除 T 细胞并确认 CAR 表达（第 5-6 天程序）。<ol><li>彻底重悬细胞以将活化的T细胞从αCD3 / CD28包被的珠子上解离（参见 材料表），并将细胞悬浮液放在磁铁上30秒。将细胞悬液转移到所需的 离体 培养容器中，并将其返回到37°C培养箱中。 注意：T细胞可以在培养瓶或深孔培养板中培养。建议在 6 孔规格板的深孔中，每孔在 30 mL mTCM 完全培养基中平板至少 5 x 106 个 T 细胞（参见 材料表）。</li>
		<li>通过流式细胞术测定 CAR 表达 8. 注意：GFP-CAR表达通过与100 ng/mL纯化的GFP孵育来测定。掺入 N 端表位标签将有助于检测替代 CAR 构建体。</li>
	</ol></li>
	<li>进行CAR-T细胞 的体外 培养（第7-10天程序）。<ol><li>在 离体 培养期间，通过去除 50% 的培养基并每 48 小时用新鲜的 mTCM-complete + 2x 10 ng/mL 的 IL-7 和 IL-15 代替来维持细胞。 注意：小鼠CAR-T细胞在活化后7天即可用于下游应用，由于解冻后活力差，不应冷冻保存。</li>
	</ol></li>
</ol>

在同基因模型中生成小鼠 CAR-T 细胞的简化方案

<ol>
	<li>June, C. H., Sadelain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+therapy.">Chimeric antigen receptor therapy.</a> N Engl J Med. 379 (1), 64-73 (2018).</li><li>Duncan, B. B., Dunbar, C. E., Ishii, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+a+clinical+lens+to+animal+models+of+car-t+cell+therapies.">Applying a clinical lens to animal models of car-t cell therapies.</a> Mol Ther Methods Clin Dev. 27, 17-31 (2022).</li><li>Hou, A. J., Chen, L. C., Chen, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigating+CAR-T+cells+through+the+solid-tumour+microenvironment.">Navigating CAR-T cells through the solid-tumour microenvironment.</a> Nat Rev Drug Discov. 20 (7), 531-550 (2021).</li><li>Campesato, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+the+ahr+restricts+a+treg-macrophage+suppressive+axis+induced+by+l-kynurenine.">Blockade of the ahr restricts a treg-macrophage suppressive axis induced by l-kynurenine.</a> Nat Commun. 11 (1), 4011 (2020).</li><li>Kaneda, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pi3kgamma+is+a+molecular+switch+that+controls+immune+suppression.">Pi3kgamma is a molecular switch that controls immune suppression.</a> Nature. 539 (7629), 437-442 (2016).</li><li>Hyrenius-Wittsten, A., Roybal, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paving+new+roads+for+cars.">Paving new roads for cars.</a> Trends Cancer. 5 (10), 583-592 (2019).</li><li>Giavridis, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR+T+cell-induced+cytokine+release+syndrome+is+mediated+by+macrophages+and+abated+by+il-1+blockade.">CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by il-1 blockade.</a> Nat Med. 24 (6), 731-738 (2018).</li><li>Lanitis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+gene+engineering+of+murine+CAR-T+cells+reveals+the+beneficial+effects+of+il-15+coexpression.">Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of il-15 coexpression.</a> J Exp Med. 218 (2), e20192203 (2021).</li><li>Lambeth, C. R., White, L. J., Johnston, R. E., De Silva, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry-based+assay+for+titrating+dengue+virus.">Flow cytometry-based assay for titrating dengue virus.</a> J Clin Microbiol. 43 (7), 3267-3272 (2005).</li><li>Agarwal, S., Wellhausen, N., Levine, B. L., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+crispr-engineered+CAR-T+cells.">Production of human crispr-engineered CAR-T cells.</a> J Vis Exp. 169, e62299 (2021).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Animal+Research.">Lab Animal Research.</a> Sterile Tissue Harvest. , (2023).</li><li>Giordano-Attianese, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computationally+designed+chimeric+antigen+receptor+provides+a+small-molecule+safety+switch+for+t-cell+therapy.">A computationally designed chimeric antigen receptor provides a small-molecule safety switch for t-cell therapy.</a> Nat Biotechnol. 38 (4), 426-432 (2020).</li><li>Kuhn, N. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cd40+ligand-modified+chimeric+antigen+receptor+T+cells+enhance+antitumor+function+by+eliciting+an+endogenous+antitumor+response.">Cd40 ligand-modified chimeric antigen receptor T cells enhance antitumor function by eliciting an endogenous antitumor response.</a> Cancer Cell. 35 (3), 473-488.e6 (2019).</li><li>Jin, C., Ma, J., Ramachandran, M., Yu, D., Essand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR+T+cells+expressing+a+bacterial+virulence+factor+trigger+potent+bystander+antitumour+responses+in+solid+cancers.">CAR T cells expressing a bacterial virulence factor trigger potent bystander antitumour responses in solid cancers.</a> Nat Biomed Eng. 6 (7), 830-841 (2022).</li><li>Kurachi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+retroviral+transduction+of+mouse+T+cells+for+in+vivo+assessment+of+gene+function.">Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function.</a> Nat Protoc. 12 (9), 1980-1998 (2017).</li><li>Jafarzadeh, L., Masoumi, E., Fallah-Mehrjardi, K., Mirzaei, H. R., Hadjati, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+persistence+of+chimeric+antigen+receptor+(CAR)+T+cell+in+adoptive+cancer+immunotherapy:+Challenges+and+ways+forward.">Prolonged persistence of chimeric antigen receptor (CAR) T cell in adoptive cancer immunotherapy: Challenges and ways forward.</a> Front Immunol. 11, 702 (2020).</li><li>Elkassar, N., Gress, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+IL-7+biology+and+its+use+in+immunotherapy.">An overview of IL-7 biology and its use in immunotherapy.</a> J Immunotoxicol. 7 (1), 1-7 (2010).</li><li>Osinalde, N., 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=Simultaneous+dissection+and+comparison+of+IL-2+and+IL-15+signaling+pathways+by+global+quantitative+phosphoproteomics.">Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics.</a> Proteomics. 15 (2-3), 520-531 (2015).</li><li>Eremenko, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+protocol+for+the+retroviral+transduction+of+mouse+CD4+T+cells.">An optimized protocol for the retroviral transduction of mouse CD4 T cells.</a> STAR Protoc. 2 (3), 100719 (2021).</li><li>Lewis, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reproducible+method+for+the+expansion+of+mouse+CD8++T+lymphocytes.">A reproducible method for the expansion of mouse CD8+ T lymphocytes.</a> J Immunol Methods. 417, 134-138 (2015).</li></ol>

没有声明利益冲突。

该方案描述了小鼠T细胞逆转录病毒转导以产生用于 体内 研究的CAR-T细胞所需的步骤和试剂。优化逆转录病毒转导条件可实现稳健的CAR表达，而无需通过超速离心或其他试剂进行病毒浓缩。但是，可以对这种方法进行多种修改。
虽然该协议描述了GFP特异性CAR的示例生成，但这些方法可以适应于生成靶向任何感兴趣的细胞外抗原的小鼠CAR-T细胞。本研究中使用的pMSCV_PGK逆?...

表达嵌合抗原受体 （CAR） 的过继性 T 细胞疗法通过利用适应性免疫系统的力量特异性靶向和消除抗原阳性癌细胞，彻底改变了癌症免疫治疗领域1。虽然靶向B细胞恶性肿瘤的CAR-T细胞疗法的成功已经得到临床验证，但在动物模型中进行的临床前研究对于开发靶向实体瘤的新型CAR仍然至关重要。然而，到目前为止，在实体瘤适应症中已证明临床疗效有限，并且越来越明显的是，单个临床前模型无法准确预测活体药物的药效学和临床疗效2,3。因此，研究人员已开始扩大CAR-T细胞产品的临床前研究，分别包括人类和小鼠癌症的异种移植和同基因模型的平行评估。
与异种移植模型不同，异种移植模型将人类肿瘤和T细胞移植到免疫缺陷小鼠中，同基因模型能够在功能免疫系统的背景下检查CAR-T细胞反应。具体来说，携带同基因肿瘤的免疫能力小鼠提供了一个系统来研究过继转移的 T 细胞与环境特异性环境之间的相互作用——包括已知抑制肿瘤微环境 （TME） 中 T 细胞功能的肿瘤相关巨噬细胞 （TAM） 和调节性 T 细胞 （Tregs）4,5,6.此外，同基因模型提供了一个额外的平台来评估靶向、肿瘤外毒性以及 CAR-T 细胞与可能导致其他毒性（包括细胞因子释放综合征）的宿主因子的相互作用 7。
尽管有这些优势，但同基因CAR-T细胞研究的数量仍然有限。值得注意的是，同基因模型需要对来自同一小鼠品系的 CAR-T 细胞进行自体工程改造，因此由于缺乏高效的小鼠 T 细胞转导和离体扩增的方法，因此提出了额外的挑战 2,8。该协议概述了通过产生逆转录病毒载体和优化的T细胞转导来实现稳定CAR表达的方法。整个过程的示意图如图1所示。这种方法的使用证明了小鼠CAR-T细胞的有效逆转录病毒转导，并且无需通过超速离心实现高CAR表达，而无需病毒浓缩。除了其他转基因的共表达外，还讨论了改变CAR构建体抗原特异性的策略。

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			<td>1 mL syringe&#160;</td>
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			<td>1.5 mL microcentrifuge tubes&#160;</td>
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			<td>10 mL syringe&#160;</td>
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			<td>100 &#956;m strainer</td>
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			<td>15 cm TC treated cell culture dishes</td>
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			<td>15 mL conical tubes&#160;</td>
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			<td>40 &#956;m strainer&#160;</td>
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			<td>50 mL conical tubes&#160;</td>
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			<td>70 &#956;m strainer</td>
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			<td>Attune NxT Flow Cytometer&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
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			<td>BALB/C, 6-8 week old&#160;</td>
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			<td>B-Mercaptoethanol&#160;</td>
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			<td>GOLDBIO</td>
			<td>A-420-500</td>
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			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS), without Calcium and Magnesium&#160;</td>
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			<td>19851</td>
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			<td>FACS buffer&#160;</td>
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			<td>Fetal bovine serum (FBS)&#160;</td>
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			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35-050-061</td>
			<td></td>
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			<td>G-Rex6</td>
			<td>Wilson Wolf</td>
			<td>80240M&#160;</td>
			<td></td>
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			<td>HEPES Buffer Solution&#160;</td>
			<td>Gibco</td>
			<td>15-630-080</td>
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			<td>Human recombinant IL-15&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-765</td>
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			<td>Human recombinant IL-2</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-748</td>
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			<td>Human recombinant IL-7</td>
			<td>Miltenyi Biotec</td>
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			<td>Lipofectamine 3000</td>
			<td>Invitrogen</td>
			<td>L3000008</td>
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			<td>MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Gibco</td>
			<td>11140-050</td>
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			<td>Mouse Anti-CD3 BV421</td>
			<td>Biolegend</td>
			<td>100228</td>
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			<td>Mouse Anti-CD3/CD28 Dynabeads</td>
			<td>Gibco</td>
			<td>11-453-D</td>
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			<td>Mouse Anti-CD4 BV605</td>
			<td>BD</td>
			<td>563151</td>
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			<td>Mouse Anti-CD44 APC&#160;</td>
			<td>Biolegend</td>
			<td>103011</td>
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			<td>Mouse Anti-CD62L PE-Cy7</td>
			<td>Tonbo</td>
			<td>SKU 60-0621-U025</td>
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			<td>Mouse Anti-CD8 APC-Cy7</td>
			<td>Tonbo</td>
			<td>SKU 25-0081-U025</td>
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			<td>Nikon Ti2 with Prime 95B camera&#160;</td>
			<td>Nikon</td>
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			<td>Non-treated 24 well plates&#160;</td>
			<td>CytoOne</td>
			<td>CC7672-7524</td>
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			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31-985-062</td>
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			<td>pCL-Eco</td>
			<td>Addgene</td>
			<td>#12371</td>
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			<td>Penicillin/Streptomycin Solution</td>
			<td>Gibco</td>
			<td>15-070-063</td>
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			<td>Phoenix Eco cells</td>
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			<td>CRL-3214</td>
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			<td>pMDG.2</td>
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			<td>#12259</td>
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			<td>Purified sfGFP</td>
			<td>N/A</td>
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			<td>RetroNectin (&#39;transduction reagent&#39;)</td>
			<td>Takara Bio</td>
			<td>T100B</td>
			<td></td>
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		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>21875</td>
			<td></td>
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			<td>Serological pipette 10 mL</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-678-11E</td>
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			<td>Serological pipette 25 mL</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-678-11</td>
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			<td>Serological pipette 5 mL</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-678-11D</td>
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			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11-360-070</td>
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			<td>TC-treated 24 well plates&#160;</td>
			<td>Corning</td>
			<td>08-772-1</td>
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		<tr>
			<td>Trypan blue&#160;</td>
			<td>Gibco</td>
			<td>15-250-061</td>
			<td></td>
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efficient generation of murine chimeric antigen receptor (car)-t cells

利用嵌合抗原受体（CAR）-T细胞的工程细胞疗法在血液系统恶性肿瘤患者中取得了显着的疗效，目前正在开发用于治疗各种实体瘤。到目前为止，新型CAR-T细胞产品的初步评估主要在使用免疫缺陷小鼠的异种移植肿瘤模型中进行。选择这种方法是为了促进人类CAR-T细胞在实验环境中的成功植入。然而，肿瘤和CAR-T细胞来源于同一小鼠品系的同基因小鼠模型允许在功能免疫系统和综合肿瘤微环境（TME）的背景下评估新的CAR技术。此处描述的方案旨在通过提供逆转录病毒转导和 离体 T细胞培养的标准化方法来简化小鼠CAR-T细胞生成过程。本协议中描述的方法可应用于本研究中使用的方法以外的其他CAR构建体，以便在免疫能力系统中对新的CAR技术进行常规评估。

这里描述的方案旨在标准化小鼠T细胞转导产生小鼠CAR-T细胞的过程。 图 1 提供了所涉及步骤的详细说明。该过程始于 通过 将病毒成分共转染到 Phoenix Eco 细胞中来生产逆转录病毒载体。 图 2 显示了转染当天 Phoenix Eco 细胞的最佳密度图像。然后，在转染后24小时或本方案的“第0天”激活分离的T细胞，为第1天的转导做准备。转导后，CAR-T细胞在重...

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

作者聚焦：增强同基因肿瘤模型中的 CAR-T 细胞功能 

该方案简化了逆转录病毒载体的产生和小鼠T细胞的转导，促进了小鼠CAR-T细胞的高效生成。

小鼠嵌合抗原受体（CAR）-T细胞的高效生成

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

我们的研究探索了工程细菌和CAR T细胞在实体瘤治疗中的有效组合。对我们来说，使用卵巢癌的同基因模型研究这两种细胞疗法如何在完整免疫系统的背景下发挥作用是很重要的。由于缺乏用于生成小鼠CAR T细胞的标准化方案，研究CAR T细胞在同基因肿瘤模型中的功能受到限制。使用传统方法在体外培养中转导小鼠T细胞特别困难。我们开发了该协议以简化小鼠CAR T细胞的生产，以便在同基因模型中进行研究，从而为评估新CAR技术的活性提供重要背景。

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

Research

JoVE Journal

Bioengineering

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

2.5K 次观看.  Columbia University. 我们的研究探索了工程细菌和CAR T细胞在实体瘤治疗中的有效组合。对我们来说，使用卵巢癌的同基因模型研究这两种细胞疗法如何在完整免疫系统的背景下发挥作用是很重要的。由于缺乏用于生成小鼠CAR T细胞的标准化方案，研究CAR T细胞在同基因肿瘤模型中的功能受到限制。使用传统方法在体外培养中转导小鼠T细胞特别困难。我们开发了该协议以简化小鼠CAR T细胞的?...

视频: 作者聚焦：增强同基因肿瘤模型中的 CAR-T 细胞功能 

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

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

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

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

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

科研

生物工程

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

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

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

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

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

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

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

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

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 ...

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

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

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

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

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

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

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

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

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

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

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

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

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

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

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

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

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

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

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

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

DNA张力探针用于绘制免疫细胞的瞬时皮牛顿受体力

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

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

从多能干细胞生成人体血管类器官

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

This method describes the encapsulation of the rabies antigen into biodegradable polymeric microparticles with structural and material properties that enable pulsatile release after a predetermined delay. Enzyme-linked immunosorbent assay (ELISA) assessment of the antigen retrieved from the particle core confirms the presence of intact trimeric rabies virus glycoprotein through particle fabrication.

包封狂犬病抗原的脉冲聚合物微粒的制备

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately ...

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.

符合GMP的基因修饰T细胞生成程序

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