Selektiv Cell Elimination fra Mixed 3D Kultur Ved hjælp af en i nærheden af infrarød Photoimmunotherapy Teknik
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
Eliminering specifikke celler uden at beskadige andre celler er yderst vanskeligt, især i etablerede væv, og der er et presserende behov for en celle eliminationsmetode i vævet ingeniørområdet. Dag inden for regenerativ medicin, vævskulturer benytter embryonale stamceller (ES), pluripotente stamceller (PSC) eller induceret pluripotente stamcelle (IPS) er lovende materialer 1 – 3 ud.
Selvom denne vævsregeneration er lovende, kontaminering med uønskede celler er et stort problem. Desuden er der en sikkerhedsrisiko for tumorgenicitet efter transplantation 4,5. Skønt mange undersøgelser har fokuseret på disse emner for at fjerne specifikke celler, især i regenerativ medicin 6 – 8, er blevet udviklet nogen praktisk metode.
I nærheden af infrarød photoimmunotherapy (NIR-PIT) er en behandling baseret på et antistof-fotoabsorberende conjugate (APC). En APC består af en celle-specifikt monoklonalt antistof (mAb) og et fotoabsorberende, IR700. IR700 er et hydrofilt silica-phthalocyanin derivat og inducerer ikke fototoksicitet af sig selv 9. IR700 er kovalent konjugeret til antistoffet via amidresterne på sidekæden af lysin molekyler. APC binder målmolekyler på cellemembranen og derefter inducerer næsten øjeblikkelig celle nekrose efter udsættelse for NIR lys ved 690 nm. Under udsættelsen for NIR-lys, cellemembranen brister fører til celle Død 9 – 14. NIR-PIT har vist sig at være effektiv med flere antistoffer eller antistoffragmenter, herunder anti-EGFR, anti-HER2, anti-PSMA, anti-CD25, anti-mesothelin, anti-GPC3, og anti-CEA 15 -. 21 Derfor kan NIR-PIT anvendes mod en bred vifte af målmolekyler. Desuden NIR-PIT er en velkontrolleret behandling, der muliggør selektiv behandling af specifikke regioner ved at begrænse NIR-light bestråling 18,22.
Her præsenteres en metode til specifik celle eliminering hjælp NIR-PIT fra blandede 3D kulturer.
Protocol
Representative Results
Discussion
Vi demonstrerer en fremgangsmåde til specifik celle eliminering fra en blandet 3D cellekultur uden beskadigelse ikke-målceller ved hjælp af NIR-PIT. Hidtil er der ingen praktisk metode til celleeliminering når vævet er etableret, eller efter transplantation. Således NIR-PIT er en lovende metode til at opnå dette. Denne teknik kan også anvendes in vivo 18,22, eftersom APC'er viser samme farmakokinetik som mAb selv. Målcelletypen kan tilpasses med forskellige APC. Forskellige anti…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev støttet af Intramural Research Program for National Institutes of Health, National Cancer Institute, Center for Cancer Research.
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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