高维流式细胞术用于解剖种植体组织的免疫功能分析
Summary
从解剖的植入物中分离细胞并通过流式细胞术对其进行表征，可以显著有助于了解针对植入物的免疫反应模式。本文描述了一种从解剖植入物中分离细胞及其染色以进行流式细胞术分析的精确方法。
Abstract
在个体体内植入实验室培养的组织或医疗设备的成功取决于受体宿主的免疫反应。将植入物视为异物，敌对和失调的免疫反应可能导致植入物的排斥反应，而调节反应和稳态的恢复可能导致其接受。分析在 体内 或 离体 环境中解剖的植入物的微环境有助于了解免疫反应的模式，这最终有助于开发新一代生物材料。流式细胞术是一种众所周知的技术，用于根据细胞表面标志物表征免疫细胞及其亚群。本综述描述了一种基于手动切割、酶消化和通过细胞过滤器过滤的方案，用于从解剖的植入物组织中分离均匀的细胞悬浮液。此外，还解释了多色流式细胞术染色方案，以及初始流式细胞仪设置的步骤，以通过流式细胞术表征和定量这些分离的细胞。
Introduction
医学领域的进步导致频繁使用植入材料来支持受损组织的功能或再生长 1,2。这些设备包括起搏器、重建美容植入物和用于骨折固定的骨科板等设备 3,4。然而，用于制造这些植入物的材料及其植入位置在决定这些植入物的成功方面起着重要作用5,6,7。作为异物，这些植入物可以产生来自宿主的免疫反应，从而导致排斥或耐受8.这一因素推动了生物材料研究，以产生能够在植入后吸引所需免疫反应的材料 9,10,11,12。
免疫反应是再生医学领域的基本要求，在再生医学领域，组织或器官在实验室的生物材料骨架（支架）周围生长，以替换受损的组织或器官13,14,15,16。在再生医学中，目标是通过使用细胞、信号和支架来替换缺失或受损的组织，其中每个细胞都可以通过免疫反应极大地调节17。此外，即使需要缺乏免疫反应，也很少缺乏免疫活性，而不是存在所需的调节谱18。流式细胞术等技术可以在表征对用于涂覆植入装置或开发组织工程支架的各种生物材料的免疫反应模式方面发挥重要作用19。
反过来，这些信息最终将有助于开发免疫系统可以很好地耐受的植入物的生物材料，或者开发可以在组织工程中发挥建设性作用的支架。正确制备用于流式细胞术分析的样品是避免通过荧光激活细胞分选进行免疫表征结果不准确的重要步骤20,21。因此，本综述提出了一种详细的方法，可用于从支架组织中分离细胞、对细胞悬液进行染色和流式细胞术分析。
Protocol
注： 图1 概述了流式细胞术方案。 1）试剂制备 准备用于稀释酶和组织培养的培养基。将 5 mL 4-（2-羟乙基）-1-哌嗪乙磺酸 （HEPES） 缓冲溶液加入 500 mL RPMI 培养基中并摇匀。将培养基储存在4°C直至进一步使用。 计算酶溶液的体积。注：酶溶液的体积是含有酶（胶原酶和DNase I）的培养基的体积，该酶在6孔板中消化切块组…
Representative Results
用于免疫分析的流式细胞术panel的开发过程通常依赖于将结果与现有数据和现场文献进行比较。了解群体在流式细胞术中的呈现方式对于正确解释数据至关重要。无论如何，群体和细胞类型在不同的组织中可能以不同的方式出现，因此可以预料到会有一些变异性。在明确定义的对照组织的背景下，可以针对具有充分研究的细胞类型的已知组织评估这种染色优化。 <strong class…
Discussion
本综述描述了一种从生物材料植入物中分离细胞以获得均匀细胞悬液的详细方法。此外，还提供了用于多色流式细胞术的细胞悬液染色的详细方案，以及配置流式细胞仪以获得最佳结果的步骤。细胞分离方法可能涉及多个步骤，通常利用手动组织解剖，然后用蛋白水解酶进行酶消化，以解离组织中的细胞外基质并破坏细胞-细胞连接，从而将单个细胞从组织中释放出来。消化后，需要进一步处理，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了美国国立卫生研究院校内研究计划的部分支持，包括国家生物医学成像和生物工程研究所。 免责声明：美国国立卫生研究院、其官员和员工不推荐或认可任何公司、产品或服务。
Materials
| 50 mL conical tubes | Fisher Scientific | 14-432-22 | |
| 6 Well Plate | Fisher Scientific | 07-000-646 | |
| BD Brilliant Stain Buffer Plus | BD Biosciences | 566385 | |
| BD Cytofix | BD Biosciences | 554655 | For only fixing cells |
| Bovine serum albumin | Millipore Sigma | A7906 | For preparing FACS staining buffer |
| CD11b AF700 | Biolegend | 101222 | Clone: M1/70 |
| CD11c PerCP/Cy5.5 | Biolegend | 117325 | Clone: N418 |
| CD197 PE/Dazzle594 | Biolegend | 120121 | Clone: 4B12 |
| CD200R3 APC | Biolegend | 142207 | Clone: Ba13 |
| CD206 PE | Biolegend | 141705 | Clone: C068C2 |
| CD45 BUV737 | BD Biosciences | 612778 | Clone: 104/A20 |
| CD86 BUV395 | BD Biosciences | 564199 | Clone: GL1 |
| CD8a BV421 | Biolegend | 100737 | Clone: 53-6.7 |
| Comp Bead anti-mouse | BD Biosciences | 552843 | For compensation control |
| DNase I | Millipore Sigma | 11284932001 | Bovine pancreatic deoxyribonuclease I (DNase I) |
| F4/80 PE/Cy7 | Biolegend | 123113 | Clone: BM8 |
| Fc Block | Biolegend | 101301 | Clone: 93 |
| Fixation/Permeabilization Solution Kit | BD Biosciences | 554714 | For fixing and permeabilization of cells. |
| HEPES buffer | Thermo Fisher | 15630080 | Buffer to supplement cell media |
| Liberase | Millipore Sigma | 5401127001 | Blend of purified Collagenase I and Collagenase II |
| LIVE/DEAD Fixable Blue Dead Cell Stain Kit | Thermo Fisher | L23105 | Viability dye |
| Ly6c AF488 | Biolegend | 128015 | Clone: HK1.4 |
| Ly6g BV510 | Biolegend | 127633 | Clone: 1A8 |
| MHCII BV786 | BD Biosciences | 742894 | Clone: M5/114.15.2 |
| Phosphate buffer saline | Thermo Fisher | D8537 | |
| RPMI | Thermo Fisher | 11875176 | Cell culture media |
| Siglec F BV605 | BD Biosciences | 740388 | Clone: E50-2440 |
| V-bottom 96-well plate |
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Cite This Article
Lokwani, R., Sadtler, K. High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues. J. Vis. Exp. (175), e61767, doi:10.3791/61767 (2021).