微流控缓冲液更换为无干扰微/纳米颗粒细胞工程
Summary
这个协议描述了使用惯性基于微流体缓冲交换策略的纯化微/纳米工程细胞与未结合的颗粒的有效消耗。
Abstract
与活性成分载微/纳米工程细胞（NPS）正在成为一个日益流行的方法，以提高本地治疗特性，使生物成像和控制细胞表型。一个关键尚未充分解决的问题是，仍无法通过常规的离心容易地除去细胞标记后未结合颗粒的显著数目。这导致增加的生物成像的背景噪声，并且可以赋予变革效应到相邻的非靶细胞。在这个协议中，我们提出称为院长流分离（DFF）惯性微流体为基础的缓冲区交换策略，以高通量的方式免费纳米粒子标记细胞有效地分离。发达螺旋微型装置便于悬浮在新的缓冲溶液纯化的细胞（THP-1和干细胞）的连续集合（> 90％细胞回收），同时实现未结合的荧光染料或染料加载纳米粒（SIL的> 95％的耗尽ICA或PLGA）。这个单步，基于大小的细胞纯化策略使高细胞处理吞吐量（10 6个细胞/分），并且是微/纳米工程细胞大容积细胞纯化以实现无干扰的临床应用是非常有用的。
Introduction
由代理加载微/纳米颗粒（纳米颗粒）的工程细胞是一种简单的，基因组整合-自由，和通用的方法，以提高生物成像能力和增加/再生医学补充其天然的治疗性质。1-3蜂窝修改都通过标记所获得的质膜或细胞质剂加载纳米粒的过量浓度以饱和结合位点。然而，这种方法的主要缺点是留在溶液中后细胞标记过程未结合颗粒的显著数量时，其可以潜在地混淆颗粒工程细胞的精确识别或4,5-复杂化的治疗效果。此外，接触含变革纳米粒在浓度过高剂（生长因子，皮质类固醇等 ）可导致细胞毒性和误导暴露可引起对非靶细胞意想不到的后果。甚至包括o颗粒载体F“生物相容性”的材料[ 例如 ，聚（乳酸共乙醇酸），PLGA]可以煽动在某些条件下，以及有效的免疫细胞反应。6这与免疫功能受损（ 例如 ，类风湿性关节炎），其潜在的延迟个体特别危险全身纳米间隙。7因此，现有的引入颗粒的工程细胞的高效去除游离颗粒是非常重要的，以减少毒性和降低误导暴露于体内剂加载颗粒。
常规梯度离心常被用来改造的细胞从游离颗粒中分离，但是费力的和在批处理模式操作。此外，通过高速离心期间细胞和密度梯度介质可能损害细胞的完整性和/或影响细胞行为的组分经历剪切应力。8微流体是与SEV一个有吸引力的替代方案ERAL分离技术，包括确定性的横向位移（DLD）9，dieletrophoresis 10,11和acoustophoresis为小颗粒分离和缓冲交换应用开发12。然而，这些方法从低通量遭受（1-10微升·分钟– 1），并容易堵塞的问题。活性分离如基于介电电泳的方法还需要在固有的介电电泳细胞表型或其他细胞标记的步骤来实现的分离的差异。一个更有前景的方法包括惯性微流体-颗粒或细胞的横向迁移跨越简化集中在由于在高雷诺数（Re）13显性升力（F L）不同的位置由于其高流动条件和优越的尺寸分辨率。 ，它经常被利用的规模为基础的细胞分离14,15和缓冲交换应用。16-18 HoweveR，缓冲液交换性能仍然很差与~10-30％污染物的溶液，作为分离的细胞通常保持接近原始和新的缓冲液之间的边界。16-18更重要的是，靶细胞的大小分布是相似的实现精确惯性聚焦和分离从其中提出了一个问题特别是在异质大小的细胞类型，如间充质干细胞（MSC）的处理的原缓冲溶液19中
我们先前已开发出一种新的惯性微流体细胞分选技术称为迪安流分离（DFF），用于使用一个2入口，2-插座螺旋微器件隔离循环肿瘤细胞（的CTC）从全血20和细菌21。在此视频协议中，我们将描述标签的THP-1（人急性单核细胞白血病细胞系）的悬浮液单核细胞（〜15微米）和干细胞（10-30微米）用钙黄绿素-L的方法oaded纳米粒，其次是DFF螺旋微型为标记的细胞和除去未纳米粒的有效回收的制造和操作。22这种单一步骤纯化策略使标记的悬浮液，悬浮于新鲜缓冲液中没有离心粘附细胞连续恢复。此外，它可以处理高达10万个细胞·毫升-1，细胞密度为顺应再生医学应用。
Protocol
Representative Results
Discussion
本文所描述的DFF细胞纯化技术使以高通量的方式标记的细胞的快速和连续分离。这种分离方法非常适合于大样本容量或高浓度的细胞样本处理，比传统的基于膜过滤是容易长时间使用后堵塞更好。同样，基于亲和力的磁选需要这是费力又昂贵的附加细胞标记的步骤。纯化的细胞被示为在通道内保持它们的标记，由于停留时间短剂和细胞形态以及（ 图3）以最小流量/剪切诱导的作用（?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Kind gift of THP-1 cells from Dr. Mark Chong and assistance in microfabrication from Dr. Yuejun Kang and Dr. Nishanth V. Menon (School of Chemical and Biomedical Engineering, Nanyang Technological University) were greatly acknowledged. This project was funded by NTU-Northwestern Institute of Nanomedicine (Nanyang Technological University). H.W.H. was supported by Lee Kong Chian School of Medicine (LKCMedicine) postdoctoral fellowship.
Materials
| Cell lines & Media | |||
| Mesenchymal Stem Cells (MSCs) | Lonza | PT-2501 | |
| Dulbecco’s modified Eagle’s medium (DMEM) | Lonza | 12-614F | |
| Fetal Bovine Serum (FBS) | Gibco | 10270-106 | |
| THP-1 monocyte cells (THP-1) | ATCC | TIB-202 | |
| Roswell Park Memorial Institute (RPMI) 1640 media | Lonza | 12-702F | |
| Name | Company | Catalog Number | Comments/Description |
| Reagents & Materials | |||
| 0.01% poly-L-lysine (PLL) | Sigma-Aldrich | P8920 | |
| 3 mL Syringe | BD | 302113 | Syringe 3ml Luer-Lock |
| 60 ml Syringe | BD | 309653 | Syringe 60ml Luer-Lock |
| Bovine Serum Albumin (BSA) | Biowest | P6154-100GR | |
| Calcein, AM (CAM) | Life Technologies | C1430 | |
| Calcein | Sigma-Aldrich | C0875 | |
| Isopropanol | Fisher Chemical | #P/7507/17 | HPLC Grade 2.5 L |
| Phosphate-Buffered Saline (PBS) | Lonza | 17-516Q/12 | |
| Plain Microscope Slides | Fisher Scientific | FIS#12-550D | 75 X 25 X 1 mm |
| Polydimethylsiloxane (PDMS) | Dow Corning | SYLGARD® 184 | |
| Scotch tape | 3M | 21200702044 | 18mm x 25m |
| Silica NPs (∼200 μm) | Sigma-Aldrich | 748161 | Pore size 4 nm |
| Syringe Tip | JEC Technology | 7018302 | 23GA .013 X .25 |
| Trypsin-EDTA (0.25%) | Life Technologies | 25200-056 | |
| Tygon Tubing | Spectra-Teknik | 06419-01 | .02X.06" 100 |
| Name | Company | Catalog Number | Comments/Description |
| Equipment | |||
| Biopsy punch | Harris Uni-Core | 69036-15 | 1.50 mm |
| Dessicator | Scienceware | 111/4 IN OD | |
| High-speed Camera | Phantom | V9.1 | |
| Inverted phase-contrast microscope | Nikon | Eclipse Ti | |
| Plasma cleaner | Harrick Plasma | PDC-002 | |
| Syringe Pump | Chemyx | CX Fusion 200 | |
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