MDA-MB-231 是 Shane Stecklein 博士的一份善意礼物。作者感谢堪萨斯大学癌症中心为进行这项研究提供资金。

注意：所有细胞培养工作均在生物安全柜中完成，并配有实验室外套、手套并遵循标准无菌技术。
1. 生成表达 CAR 的 Jurkats （CAR-J）
<ol><li>购买 Jurkat 细胞，从 ATCC 克隆 E6-1。在 T-75 烧瓶中解冻 1 x 106 个细胞，并在 T-75 烧瓶中培养。使用补充有10%FBS的Roswell Park Memorial Institute（RPMI）培养基在37°C和5%CO2的培养箱中，以0.6×106个细胞/mL的悬浮液维持它们。</li>
	<li>培养板 1 x 10 5 个 Jurkat 细胞，每孔组织培养物处理 24 孔板，在含有 4 μg/mL 聚凝胺的 500 μL RPMI 生长培养基中，可提高慢病毒效率。使用基于激光的荧光检测台式细胞分析仪对细胞进行计数。孔的数量取决于要评估的结构数量。此示例将使用 4 个构造和一个未转导的控件。CAR结构设计如表1所示。</li>
	<li>每个CAR构建体中加入10μL慢病毒/孔。CAR构建体设计和慢病毒生产如前所述12。</li>
	<li>第二天，向每个孔中加入1mL生长RPMI培养基，并继续在37°C的培养箱中用5%CO2培养。</li>
	<li>2 天后（Jurkat 转导后总共 72 小时）收集细胞并计数。</li>
	<li>取 1 x 104 个细胞进行流动，以确认 Jurkat 细胞上的 CAR 表达。简而言之，用FACS染色溶液（FSS）洗涤细胞2次，然后用靶向用于检测CAR阳性的抗体标记CAR-J，用于在4°C的黑暗中检测CAR阳性30分钟。 用FSS再次洗涤2x，并如前所述通过流式细胞仪运行细胞12。按照制造商的建议使用抗体浓度。</li>
	<li>Jurkat 细胞易于转导，几乎总是显示 >90% 的 CAR 表达。在共培养细胞毒性测定之前很久就生产 CAR-J 并冷冻以备后用。</li>
</ol>2.铺板CFSE标记的肿瘤细胞
注意：MDA-MB-231（来自ATCC，HTB-26）细胞是合作者的礼物，EGFR KO MDA-MB-231如前所述12。
<ol><li>在 T-75 烧瓶中解冻 1 x 106 个细胞，并在 T-75 烧瓶中培养。将MDA-MB-231肿瘤细胞和CRISPR EGFR KO MDA-MB-231肿瘤细胞维持在14mL补充有10%胎牛血清（FBS）的Dulbecco改良鹰培养基（DMEM）中，在37°C的培养箱中用5%CO2 进行，并在约70%汇合时分裂。</li>
	<li>在常规明场显微镜下观察贴壁肿瘤细胞，以确保近70%的汇合。</li>
	<li>使用血清移液管从烧瓶中取出生长培养基。向T75烧瓶中加入3mL胰蛋白酶，并置于37°C的5%CO2 培养箱中3-5分钟，使细胞从烧瓶中分离。</li>
	<li>使用等量（3mL）生长培养基中和胰蛋白酶。将细胞悬液收集在离心管中，并以400× g 旋转细胞4分钟以沉淀细胞。</li>
	<li>使用移液管弃去上清液，并将细胞重悬于2mL磷酸盐缓冲盐水（PBS）中。使用细胞计数器测定细胞浓度。</li>
	<li>将 8 x 105 个细胞转移到另一个 15 mL 试管中，并加入 PBS 使其体积为 1 mL。</li>
	<li>向每个试管中加入 2 μL 羧基荧光素琥珀酰亚胺酯（CFSE;5 μM 储备浓度），并使用 1 mL 移液管充分混合。</li>
	<li>将细胞与CFSE在37°C的培养箱中用5%CO2孵育20分钟。20分钟后从培养箱中取出试管，并向试管中加入5mL生长培养基。</li>
	<li>将试管以400× g 离心4分钟，以沉淀CFSE标记的细胞。使用移液管弃去上清液，并加入 1 mL 新鲜培养基以重悬细胞。</li>
	<li>使用细胞计数器重新评估细胞浓度。将 4 x 105 个细胞转移到 25 mL 试剂储液槽中，并加入培养基，总体积为 8 mL。<ol><li>为确保在100μL培养基中接种5000个肿瘤细胞/孔的足够体积，体积需要能够使用多通道移液器移液，并补偿步骤1.7中少数细胞的损失，准备10%-20%的额外细胞和培养基体积。</li>
	</ol></li>
	<li>使用 5 mL 血清移液管彻底混合细胞悬液。使用100 μL多通道移液管，将100 μL细胞悬液移液到每排透明平底黑色96孔板的左半部分。样品电镀策略见 表2。</li>
	<li>同样在板的右半部分移液EGFR KO细胞。将整个板铺好后，在组织培养罩平台上来回和左右拖动板，以确保肿瘤细胞在孔中的均匀分布。</li>
	<li>将板在37°C的培养箱中用5%CO2 孵育4小时，以便肿瘤细胞附着。</li>
</ol>3. 共培养表达 CAR 的 Jurkats 与 CFSE 标记的肿瘤细胞
<ol><li>使用未转导和表达 CAR 的 Jurkat 细胞的计数，将每个 CAR-J 的 4 x 105 个细胞转移到 25 mL 储液槽中。加入 DMEM 生长培养基，总体积为 2 mL。</li>
	<li>对于 4：1 的 E：T，每孔在 100 μL 培养基中加入 2 x 104 个 CAR-J，使用多通道移液管沿着每个孔的侧面轻轻移动，以免干扰附着的肿瘤细胞。</li>
	<li>使用多通道移液器将另外 100 μL 生长培养基添加到含有肿瘤细胞和 Jurkat 细胞的孔的一侧。仅肿瘤组获得 200 μL 培养基，使其在所有孔中总共获得 300 μL 培养基。</li>
	<li>沿着平台来回和左右运动拖动板，以确保 Jurkat 细胞在肿瘤细胞上的均匀分布。</li>
	<li>在37°C的培养箱中用5%CO2 共培养48小时。</li>
</ol>4. 成像板的制备
<ol><li>根据孔数在低背景荧光培养基中以 1 μg/mL 制备碘化丙啶 （PI） 溶液，每个孔获得 100 μL 培养基。</li>
	<li>对于 84 个孔，制备 9 mL 含有 PI 的培养基。使用移液管彻底混合培养基。</li>
	<li>用去离子水稀释 Triton-X 制备 10 mL 20% Triton-X 溶液。共培养48小时后，通过单次倒置板并在纸巾上敲击来弃去含有CAR-J的上清液。</li>
	<li>现在，使用多通道移液管将100μL上述制备的含有PI的培养基（步骤4.2）轻轻加入每个孔中，以免干扰粘附的肿瘤细胞。</li>
	<li>将 20 μL 20% Triton-X 溶液加入每种肿瘤类型的第一个孔中，作为完全死亡对照。</li>
	<li>将板留在培养箱中20分钟。使用荧光成像流式细胞仪对板进行成像。数据存储在计算机上，以后可以进行分析。</li>
</ol>5. 分析荧光图像
<ol><li>使用肿瘤细胞中的一个孔来设置绿色荧光通道。</li>
	<li>将孔掩模降低到 98%，以去除孔边缘的细胞，因为边缘的信号不完美。</li>
	<li>在绿色荧光 CFSE 通道上识别 CFSE 标记的肿瘤细胞。修改荧光强度阈值以拾取孔上的所有细胞。</li>
	<li>将最小细胞直径设置为25μm，以去除在CFSE通道上检测到的任何碎片。这取决于所分析的细胞类型。</li>
	<li>启用 单独的触摸对象 ，以在单个单元格相互接触时对其进行识别。</li>
	<li>在图像显示屏上选择 CFSE ，然后查看图形叠加，以确定系统正在选取的内容。</li>
	<li>一旦正确拾取CFSE标记的细胞，设置门来定义活细胞群和死细胞群。</li>
	<li>选择 CFSE 标记的单元格，生成 y 轴上的计数直方图与 x 轴上的平均 PI 强度。</li>
	<li>根据我们添加Triton-X的孔（步骤4.5），绘制一个分离器来区分低PI染色细胞和高PI染色细胞。该孔应将大部分细胞置于高 PI 染色区域。 注意：PI 显示活细胞上的基础信号。因此，添加Triton-X会杀死所有细胞，并且它们在红色荧光通道中染色明亮。这有助于绘制门以将死细胞与活细胞分离。</li>
	<li>调整 x 轴值以便能够更好地查看单元格。现在将低 PI 染色细胞标记为活细胞。</li>
	<li>选择“活动单元格”，设置另一个直方图，其面积位于 x 轴上，计数在 y 轴上。</li>
	<li>使用肿瘤绘制另一个分裂器，仅捕获细胞而不是碎片。Jurkat 处理过的井将开始积聚碎屑，这些碎屑将被 CFSE 染色，需要从计数中清除。其余的非碎片被标记为“大细胞”。</li>
	<li>对整个板运行分析，并导出包含数字的电子表格。</li>
	<li>绘制大计数图以确定暴露于 CAR-J 后孔中剩余的活 CFSE 标记肿瘤细胞的数量。使用单因素方差分析确定统计量。</li>
</ol>

评估表达 CAR 的 Jurkat 细胞对肿瘤细胞的定量杀伤

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作者没有什么可透露的。

在这里，我们提出了一种快速方法来有效地评估 Jurkat 细胞中 CAR 表达诱导的靶标特异性细胞溶解活性。所有 CAR 构建体具有相同的 scFv，但不同的铰链和跨膜结构域，这些结构域已被证明会影响 CAR-T 细胞的效力13。通过用抗原敲除 （KO） 细胞培养这些 CAR-J 来进一步评估这些 CAR-J 的非特异性杀伤。这表明杀伤是肿瘤抗原特异性的，而不是由于 CAR-J 的基础激活。可以很容易地确定?...

据美国国家癌症研究所1 报道，CAR-T 细胞疗法在血液系统恶性肿瘤中显示出巨大的前景，自 2017 年以来，FDA 批准了 6 种 CAR-T 产品。有许多CAR-T细胞在临床试验中用于靶向实体瘤。设计新的CAR靶点和优化CAR结构对于CAR-T细胞的功效至关重要。为每种应用选择理想的 CAR 构建体对于准确靶向肿瘤相关抗原 （TAA） 至关重要，同时避免正常组织中 TAA 表达水平低2。
CAR 构建体主要由五个区室组成：（1） 靶向肿瘤抗原的细胞外单链可变片段 （scFv） 结构域;（2）铰链域;（3）跨膜结构域;（4）细胞内细胞质T细胞共刺激结构域;（5）信令域。修改这些结构域中的每一个都会影响 CAR-T 细胞与其靶细胞结合的精度 3。因此，评估这些CAR构建体的细胞毒性和交叉反应性对于选择正确的构建体进行体内实验至关重要。目前评估 T 细胞溶解的方法包括 51Cr 释放试验、乳酸脱氢酶释放试验、生物发光成像试验、基于实时阻抗的细胞分析和基于细胞的流式细胞术试验 4,5。这里描述的基于荧光成像的平台可识别活细胞与死细胞的数量，这是对 T 细胞溶解的直接定量，而不是评估 T 细胞溶解的间接方法。
这是一种简单、经济高效、快速且高通量的技术，只需最少的干预，即可评估表达表皮生长因子受体 （EGFR） CAR 的 Jurkat 细胞对 MDA-MB-231 三阴性乳腺癌 （TNBC） 细胞和 EGFR CRISPR 敲除 MDA-MB-231 细胞的细胞毒性。Jurkat 细胞是永生化的人 T 淋巴细胞6，已被广泛用于研究 T 细胞活化和信号转导机制7。此外，Jurkat 细胞已在多项研究8、9、10、11 中用于体外 CAR 测试。Jurkat 细胞很容易被慢病毒转导并具有持续的增殖，该系统被用于优化各种 EGFR CAR 构建体的铰链结构域。
该测定可用于筛选靶向各种肿瘤抗原的多种CAR构建体，并用于对抗多种贴壁肿瘤细胞系和各种效应子与肿瘤（E：T）比率。此外，可以评估多个时间点，并且可以修改重复次数，以确定各种CAR构建体中的最佳杀伤。需要使用外周血单核细胞 （PBMC） 衍生的 CD3 T 细胞来确认最佳构建体。开发这种方法的总体目标是以高通量方式快速优化CAR铰链几何形状，克服转导效率低等障碍，然后在PBMC衍生的T细胞中得到确认。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Conical Tube (Sterile)</td>
			<td>Midwest Scientific</td>
			<td>#C15B</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>50 mL Conical Tube (Sterile)</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Black/Clear 96 well plate</td>
			<td>Falcon</td>
			<td>353219</td>
			<td>Celligo has a list of compatible plates</td>
		</tr>
		<tr>
			<td>Celigo 4 Channel Imaging Cytomenter</td>
			<td>Nexcelcom Bioscience</td>
			<td>200-BFFL-5C</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Celigo Software</td>
			<td>Nexcelcom Bioscience</td>
			<td>Version 5.3.0.0</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Cell Culture Incubator</td>
			<td>Thermo Scientific</td>
			<td>HeraCell 160i</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Cell Culture Treated Flasks (T75, various sizes, Sterile)</td>
			<td>TPP</td>
			<td>90076</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Tonbo</td>
			<td>13-0850-U500</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Cytek Muse Cell Analyzer</td>
			<td>Cytek</td>
			<td>0500-3115</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-040</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini bio-products</td>
			<td>900-108</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>Cytek, BD, etc</td>
			<td>Aurora, LSR II, etc</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>FlowJo Sortware</td>
			<td>Becton Dickinson &#38; Company&#160;</td>
			<td>Version 10.7.1</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Fluorobrite DMEM</td>
			<td>Gibco</td>
			<td>A18967-01</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>GraphPad Software</td>
			<td>GraphPad</td>
			<td>Version 9.3.1 (471)</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Multichanel Pipette</td>
			<td>Thermo Scientific</td>
			<td>Finnpipette F2</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>PenStrep</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Pipette tips (Sterile, filtered, 1 mL, Various sizes)</td>
			<td>Pr1ma</td>
			<td>PR-1250RK-FL, etc</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Pipettors&#160;</td>
			<td>Thermo Scientific</td>
			<td>Finnpipette F2</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Corning</td>
			<td>10-041-cv</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Serological Pipette Aid</td>
			<td>Drummond Scientific</td>
			<td>4-000-105</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Serological Pipettes (Sterile, various sizes)</td>
			<td>Pr1ma</td>
			<td>PR-SERO-25, etc</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Corning</td>
			<td>25-000-CI</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Sterile Reservoirs</td>
			<td>Midwest Scientific</td>
			<td>RESE-2000</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Any similar will work</td>
		</tr>
	</tbody>
</table>

rapid in vitro cytotoxicity evaluation of jurkat expressing chimeric antigen receptor using fluorescent imaging

嵌合抗原受体 （CAR） T 细胞处于肿瘤学的最前沿。CAR 由靶向结构域（通常是单链可变片段 scFv）构建，伴随链内连接子，然后是铰链、跨膜和共刺激结构域。链内连接子和铰链结构域的修饰可以对CAR介导的杀伤产生显着影响。考虑到 CAR 结构的每个部分都有许多不同的选项，因此有大量的排列。制造CAR-T细胞是一个耗时且昂贵的过程，制造和测试许多构建体是一项大量的时间和材料投资。该协议描述了一个平台，用于快速评估 Jurkat 细胞 （CAR-J） 中铰链优化的 CAR 构建体。Jurkat 细胞是一种永生化的 T 细胞系，具有高慢病毒摄取量，可实现高效的 CAR 转导。在这里，我们提出了一个使用荧光成像仪快速评估 CAR-J 的平台，然后确认 PBMC 衍生的 T 细胞的细胞溶解。

CAR-T cell therapy has shown great promise in hematological malignancies, evident from the 6 FDA-approved CAR-T products since 2017, as reported by the National Cancer Institute1. There are numerous CAR-T cells in clinical trials for targeting solid tumors. Engineering novel CAR targets and optimizing the CAR construct is vital to the efficacy of a CAR-T cell. Choosing the ideal CAR construct for each application is essential for accurate targeting of tumor associated antigens (TAA) while avoiding low levels of TAA expression in normal tissues2.
A CAR construct is primarily made of five compartments: (1) extracellular single-chain variable fragment (scFv) domain targeting tumor antigen; (2) hinge domain; (3) transmembrane domain; (4) intracellular cytoplasmic T cell costimulatory domain; and (5) signaling domain. Modifying each of these domains affects the precision of the CAR-T cell engaging with its target cell3. Hence, evaluating the cytotoxicity and cross-reactivity of these CAR constructs in vitro is critical to choose the right construct for progressing toward in vivo experiments. Current methods of evaluating cytolysis by T cells include 51Cr release assay, lactate dehydrogenase release assay, bioluminescent imaging assay, real-time impedance-based cell analysis, and cell-based flow cytometry assay4,5. The fluorescent imaging-based platform described here identifies the number of live vs. dead cells, which is a direct quantification of T cell cytolysis as opposed to an indirect method of evaluating the cytolysis by T cells.
Here is an easy, cost-efficient, rapid, and high throughput technique with minimal intervention to evaluate the cytotoxicity of Jurkat cells expressing epidermal growth factor receptor (EGFR) CAR against MDA-MB-231 triple-negative breast cancer (TNBC) cells and EGFR CRISPR knock out MDA-MB-231 cells. Jurkat cells are immortalized human T Lymphocyte Cells6 that have been widely used for studying T cell activation and signaling mechanisms7. Furthermore, Jurkat cells have been used for in vitro CAR testing in multiple studies8,9,10,11. Jurkat cells are easily transduced by lentivirus and have sustained proliferation, and this system was leveraged to optimize the hinge domain of various EGFR CAR constructs.
This assay can be used for screening multiple CAR constructs targeting various tumor antigens and used against multiple adherent tumor cell lines and in various effector to tumor (E:T) ratios. Additionally, multiple time points can be evaluated, and number of replicates can be modified to identify best killing among the various CAR constructs. The best constructs need to be confirmed using peripheral blood mononuclear cells (PBMCs) derived CD3 T cells. The overall goal behind developing this method is to rapidly optimize CAR hinge geometry in a high throughput manner overcoming barriers such as low transduction efficiency, followed by confirmation in PBMC derived T cells.

作者聚焦：CAR T 细胞构建体的高通量筛选以增强细胞毒性和免疫记忆 

使用荧光成像快速体外细胞毒性评估表达嵌合抗原受体的 Jurkat

CAR T-cells are being constantly improved to achieve better responses in human clinical trials. We are identifying ways to evaluate this in our lab using an unbiased high throughput method, such as using fluorescent imaging to screen combinations that work best. There are several combinations of constructs that can be designed for any single chain variable fragment by modifying the geometry of the extracellular and hinge domains.We have designed this protocol to identify the combinations with the best efficacy in eliminating target cells using Jurkat cells. This is a rapid, high throughput protocol to screen CAR constructs based on its target cell cytotoxicity using Jurkat cells, which are then validated with conventional T-cells. A dozen different CAR constructs can be tested in a 96-well plate simultaneously using this technique.Here in Akhavan Lab, we quantify in absolute numbers the direct cyclosis of target cells by the CAR expressing Jurkat cells in a high throughput manner to screen multiple CAR constructs. Our laboratory's primary aim is to improve the clinical translation of CAR T-cell therapy against high grade brain tumors. We leverage high throughput drug screens as one modality to improve CAR T-cell cytotoxicity, as well as targeting the tumor microenvironment.

在 72 小时时评估了 CAR-J1 的 E：T 比值范围在 1：8 和 8：1 之间，靶向 TNBC MDA-MB-231 细胞上的 EGFR。用含有聚凝胺的CAR慢病毒转导Jurkat细胞以产生CAR-J细胞，如步骤2所述。CAR-J1的细胞毒性随着E：T比值的增加而显著增加，而在1：8的比例下杀伤率没有差异（图1）。在 72 小时内以 4：1 E：T 观察到超过 50% 的杀伤率。该 E：T 用于后续实验，持续时间缩短至 48 小时，用于对多种 CAR ?...

Watch this Scientific Journal Video about High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory at JoVE.com

一种评估靶向单个肿瘤抗原的表达嵌合抗原受体 （CAR） 的 Jurkat 细胞定量肿瘤细胞杀伤的方案。该方案可用作筛选平台，用于在外周血来源的T细胞中确认之前快速优化CAR铰链结构。

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...

CAR T细胞正在不断改进，以在人体临床试验中实现更好的反应。我们正在确定在实验室中使用无偏高通量方法进行评估的方法，例如使用荧光成像来筛选最有效的组合。通过修改细胞外和铰链结构域的几何形状，可以为任何单链可变片段设计几种构建体组合。我们设计了这个方案，以确定使用Jurkat细胞消除靶细胞最有效的组合。这是一种快速、高通量的方案，使用 Jurkat 细胞根据其靶细胞细胞毒性筛选 CAR 构建体，然后用常规 T 细胞进行验证。使用这种技术可以在 96 孔板中同时测试十几种不同的 CAR 构建体。在 Akhavan 实验室，我们以绝对数量量化表达 CAR 的 Jurkat 细胞以高通量方式对靶细胞的直接环化，以筛选多种 CAR 构建体。我们实验室的主要目标是改善CAR T细胞疗法针对高级别脑肿瘤的临床转化。我们利用高通量药物筛选作为一种方式来改善CAR T细胞的细胞毒性，并靶向肿瘤微环境。

rapid-vitro-cytotoxicity-evaluation-jurkat-expressing-chimeric

Research

JoVE Journal

Cancer Research

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

1.6K 次观看.  University of Kansas Cancer Center. CAR T细胞正在不断改进，以在人体临床试验中实现更好的反应。我们正在确定在实验室中使用无偏高通量方法进行评估的方法，例如使用荧光成像来筛选最有效的组合。通过修改细胞外和铰链结构域的几何形状，可以为任何单链可变片段设计几种构建体组合。我们设计了这个方案，以确定使用Jurkat细胞消除靶细胞最有效的组合。这是一种快速、高通量的方案，使用 Jurkat ...

视频: 作者聚焦：CAR T 细胞构建体的高通量筛选以增强细胞毒性和免疫记忆 

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

科研

癌症研究

Plating CFSE-Labelled Tumor Cells and Co-Culturing CAR-Expressing Jurkats for In Vitro Cytotoxicity Evaluation

To begin using a serological pipette, remove the growth media from the T75 flask containing 70%confluent MDA-MB-231 culture. Add three milliliters of trypsin to the flask and incubate at 37 degrees with 5%carbon dioxide for three to five minutes to detach the cells from the flask. Then to neutralize the trypsin, add three milliliters of DMEM into the flask.Collect the cell suspension in a centrifuge tube and spin at 400 G for four minutes. Discard the supernatant using a pipette and resuspend the pellet in two milliliters of PBS. Using a cell counter, determine the cell concentration.Transfer eight times 10 to the fifth cells into a 15 milliliter tube and add PBS to make the volume to one milliliter. Then add two microliters of CFSE into the tube and mix the cell suspension thoroughly using a one milliliter pipette. Place the tube at 37 degrees Celsius and 5%carbon dioxide incubator for 20 minutes.Next, add five milliliters of DMEM to the tube and centrifuge to pellet down the CFSE labeled cells. Discard the supernatant and resuspend the pellet in one milliliter of DMEM. After reevaluating the cell concentration, transfer four times 10 to the fifth cells into a 25 milliliter reagent reservoir.Add DMEM to make the volume eight milliliters and mix the cell suspension with a five milliliter serological pipette. Using a multichannel pipette, transfer 100 microliters of cell suspension into each row on the left half of a black 96-well plate. Similarly, add EGFR knockout cell suspension on the right half of the plate.To ensure uniform cell distribution, move the plate back and forth and side to side on the platform. Incubate the plate for four hours at 37 degrees Celsius and 5%carbon dioxide for the tumor cells to attach. Based on counts of untransduced and CAR-expressing Jurkat cells, transfer four times 10 to the fifth CAR-J cells into a 25 milliliter reservoir.Add DMEM to the reservoir to a final volume of two milliliters. For an effector to tumor ratio of 4:1, add two times 10 to the fourth CAR-J cells per well in 100 microliters of medium along the side of the well without disturbing the attached tumor cells. Then add 100 microliters of DMEM to the side of the wells containing tumor and Jurkat cells to get 300 microliters of volume per well.Move the plate as demonstrated to ensure uniform distribution of the Jurkat cells on the tumor cells. Allow the co-culture to establish at 37 degrees Celsius with 5%carbon dioxide for 48 hours.

接种 CFSE 标记的肿瘤细胞并共培养表达 CAR 的 Jurkats 进行体外细胞毒性评估

Preparation of Plate for Fluorescent Imaging and Cell Viability Assessment in CAR-Jurkat Co-Culture with CFSE-Labeled Tumor Cells

After co-culturing CAR expressing jurkats with CFSE-labeled tumor cells, gently invert the plate on a paper towel and tap to discard the supernatants containing CAR-J. Using a multichannel pipette, add 100 microliters of FluoroBrite DMEM media containing propidium iodine, or PI, into the wells without disturbing the adhered tumor cells. Then add 20 microliters of 20%Triton X to the first well of each tumor type, serving as a total dead control.Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 20 minutes before imaging. After fluorescent imaging, use one of the tumor-only wells of cells to calibrate the green fluorescent channel. To exclude cells at the edge of the wells decrease the well mask to 98%Identify CFSE-labeled tumor cells on the green fluorescent channel and adjust the fluorescence intensity threshold value to capture all the cells in the well.Set the minimum cell diameter to 25 micrometers to exclude debris detected in the CFSE channel. Then enable separate touching objects to identify individual cells. Next set gates to define live versus dead cell populations.Select the CFSE-labeled cells and generate a histogram of counts on the y-axis versus mean PI intensity on the x-axis. Adjust the x-axis value to better visualize the cells and draw a splitter to differentiate between low PI stained cells and high PI stained cells. Label the low PI stained cells as live cells.Next, set another histogram on live cells with area on the x-axis and counts on the y-axis. Label the non-debris cells as big live cells. Then draw another splitter using the tumor only well to capture cells and filter out debris.After running the analysis on the entire plate, export the spreadsheet containing the numbers. Plot the counts of big cells to determine the number of live CFSE-labeled tumor cells remaining in the well after exposure to CAR-J. Finally perform statistical analysis using one-way ANOVA.The cytotoxicity of CAR-J1 increased with a higher effector to tumor ratio and more than 50%killing was observed at an effector to tumor ratio of four to one. EGFR-targeting CAR constructs with modified hinge domains showed antigen-specific killing against EGFR expressing MDA-MB-231 cells, and no significant killing was observed against EGFR knockout cells. CAR constructs were also expressed on PBMC derived CD3 T cells.However, there was no expression of CD8 hinge-containing EGFR-CAR. The IgG short showed the least cytotoxic potency against MDA-MB-231 cells compared to the IgG long and IgG medium.