从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除
Summary
Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).
Abstract
Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.
Introduction
消除特定细胞而不损害其他细胞是非常困难的，特别是在建立组织，并且存在用于在组织工程领域中的细胞消除方法的迫切需要。现今在再生医学领域中，使用胚胎干细胞（ES），多能干细胞（的PSCs），或诱导的多能干细胞（IPS）的组织培养物是有前途的材料1 – 3。
虽然这个组织再生是有前途的，具有不需要的细胞污染是一个大问题。此外，有移植4,5-之后致瘤性的安全问题。虽然许多研究都集中在这些问题上，以消除特定细胞，尤其是在再生医学6 – 8，没有实际的方法已被开发出来。
近红外photoimmunotherapy（NIR-PIT）是一种基于抗体 – 光吸收剂conjugat的处理E（APC）。一个APC由细胞特异性单克隆抗体（mAb）和光吸收剂，IR700的。 IR700是亲水性二氧化硅的酞菁衍生物和本身并不9诱发光毒性。 IR700是通过赖氨酸分子的侧链酰胺残基共价结合到抗体上。 APC的结合细胞膜上的靶分子，然后诱导暴露于NIR光在690纳米的近后立即细胞坏死。在暴露于近红外光，细胞膜破裂，导致细胞死亡9 – 14。 NIR-PIT已被证明是具有多个抗体或抗体片段，其中包括抗EGFR，抗HER2，抗PSMA，抗CD25，抗间皮素，抗GPC3，和抗CEA 15有效– 21。因此，近红外PIT可对多种靶分子使用。此外，近红外PIT是良好控制的治疗，通过限制近红外二极管灯允许特定区域的选择性治疗ŧ照射18,22。
这里，我们目前采用NIR-PIT从混合3D文化特异性细胞清除的方法。
Protocol
Representative Results
Discussion
我们证明特定细胞消除从混合三维细胞培养物的方法，而不使用近红外-PIT损害对非靶细胞。到目前为止，存在一旦组织被建立或细胞消除无实际方法移植后。因此，NIR-PIT是实现这个很有前途的方法。这种技术也可在体内 18,22利用，因为的APCs显示出类似的药代动力学为单克隆抗体本身。靶细胞类型可适于与各种的APC。各种抗体或抗体片段，其中包括抗EGFR，抗PSMA，抗间皮素和…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国国立卫生研究院院内研究计划，国家癌症研究所，癌症研究中心的支持。
Materials
| IRDye 700DX Ester Infrared Dye | LI-COR Bioscience (Lincoln, NE, USA) | 929-70011 | |
| Na2HPO4 | SIGMA-ALDRICH (St. Louis, MO, USA) | S9763 | |
| Sephadex G25 column (PD-10) | GE Healthcare (Piscataway, NJ, USA) | 17-0851-01 | |
| Coomassie (bradford) Plus protein assay | Thermo Fisher Scientific Inc (Waltham, MA, USA) | PI-23200 | |
| Perfecta3D 96-Well hanging Drop Plates | 3D Biomatrix Inc (Ann Arbor, MI, USA) | HDP1096-8 | |
| Optical power meter | Thorlabs (Newton, NJ, USA) | PM100 | |
| LED: L690-66-60 | Marubeni America Co. (Santa Clara, CA, USA) | L690-66-60 | |
| Vectibix (panitumumab) | Amgen (Thousand Oaks, CA, USA) | ||
| 35mm glass bottom dish, dish size 35mm, well size 10mm | Cellvis (Mountain View, CA, USA) | D35-10-0-N |
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