유리 표면 기능화를 통해 표적 세포 격리 방법
Summary
This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.
Abstract
One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.
Introduction
현재 벤치 탑 세포 분리 방법 (예, 형광 활성화 셀이 1 정렬, 레이저 캡처 마이크로 해부 (2), 면역 자기 비드 분리 1) 준비 및 정렬의 몇 시간이 걸릴 수 있습니다. 이 큰 시간 규모는 생리적 반응 (3)의 대표하지 분석 결과, 생리적 반응과 표현 수준에 영향을 줄 수 있습니다. 시스템은 신속하고 효율적으로 생체 의학 애플리케이션에 세포 분리 및 농축을 향상시키기 위해 세포 표면 수용체 수준을 중단없이 특정 세포 유형을 분리 할 수있는 요구된다. 따라서, 우리의 접근 방식에 대한 이론적 근거는 세포 분리를위한 부드러운 접근 방식을 개발하는 것입니다.
은 "칩에 연구소는"개념은 크기 빨리 (시간 – 투 – 분) 세포 분리의 주문 약속을 제공하며, 가장 자주 표면에 세포를 캡처하고 세포 또는 세포 내 콩트를 방출 포함물리 4,5를 통해 국세청 또는 화학적 방법 6. 이러한 방법은 체외 배양 (12, 13)에 대한 세포를 제공하는 경우에도 RNA 발현 9-11, 식별, 단백질 7,8 식을 식별하거나 몇 가지 장점을 제공하지만, 이러한 기술의 대부분은 이러한 세포 수용체 프로파일로 인해에게로 진단로 번역 할 수 없습니다 자신의 비 생리 학적 환경. 이러한 정확한 생리 학적 데이터를 생성하지 않습니다이 해제 에이전트를 사용하여 세포 수용체의 정량화 기법을 의미 이러한 수용체 수량 14, 15에 영향을 미칠 수있는 콜라게나, 같은 효소 리프팅 에이전트. 세포 용해는 기본 표면 수용체 사이의 분화를 방지하고 이전 16 내재화 된 것과. 이 프로토콜은 세포 분리를위한 빠르고 부드러운 접근 방식을 설명합니다.
Protocol
Representative Results
Discussion
세포 분리 기술의 향상은 혈관 생물학 (19)의 세포 재생 생물학 프로그래밍 및 혈관 신생 신호 줄기, 신경 과학 (18)의 구조 – 기능 관계의 과학적 연구를 발전한다. 실제로, 일차 세포 배양 물 (20)은 (예 된 HUVEC), 혈관 생물학 주로 세포 분리 기술의 사용을 통해 수행된다. 세포 격리는 최근 세포막 수용체 3,14,15,19,21 정량적 흐름 (qFlow) 계측법 분석에 사용 하였?…
Declarações
The authors have nothing to disclose.
Acknowledgements
We would like to thank the American Cancer Society, Illinois Division (282802) and the National Science Foundation CBET (1512598) for funding support. We also would like to thank Dr. Dianwen Zhang from the University of Illinois Beckman Institute for microscopy training. Finally, we would like to thank Jared Weddell, Stacie Chen, and Spencer Mamer for insightful discussions.
Materials
| (3-Aminopropyl) triethoxysilane (APTES) | Acros Organics | 919-30-2 | Used to make 2% APTES solution |
| Plasma Cleaner Pico | Diener | Model 1 | Cleans surfaces and allows for bonding of PDMS to glass |
| d-Desthiobiotin (DSB) | Sigma | D20655 | Used as the releasing mechanism in the cellular capture surface. |
| dimethyl sulfoxide (DMSO) | British Drug Houses (BDH) | BDH1115-1LP | Dissolves the DSB into solution |
| 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) | Thermo-Scientific | 5g: 22980 25g: 22981 |
Activates Carboxylic Acids and allows binding of proteins to glass surface. |
| uncoated 8-well culture slide | BD Falcon | Case of 24: 354118 Case of 96: 354108 |
Used in cellular experiments involving Zeiss fluorescence microscope such as initial capture and release quantification experiments |
| Glass bottom 24-well plates | MatTek | P24G-0-13-F | Used in cellular experiments involving the plate reader such as antibody and cellular titration experiments |
| Mercaptoethanol | Science Lab | 60-24-2 | Used to quench reaction between EDC and DSB |
| 4-Morpholinoethanesulfonic acid hydrate (MES Hydrate 99%) |
Fisher Scientific | AC172590250 | Used to make 0.1 M MES Buffer for use in EDC reaction |
| Precision Oven | Thermo Scientific | 11-475-153 | Used in curing of PDMS and APTES layer. |
| Titramax 1000 Shaker | Heidolph | 13-889-420 | Used to ensure even distribution of APTES on surfaces. |
| 1X Streptavidin 5mg [e7105-5mg] |
Proteo Chem | 9013-20-1 | Biotin-binding protein May cause irritation |
| 5 cm Glass Dish | Fisher Scientific | 08748A | Used in HUVEC studies as well as future profiling studies. |
| 14 cm Petri Dish with Cover | Sigma-Aldrich | Z717231 | Used to hold samples being functionalized and transport them. |
| MCF7-GFP cells | Cell Biolabs | AKR211 | Stored in liquid nitrogen |
| RAW264.7 mouse macrophages |
ATCC | TIB-71 | Gifted to us from Smith lab at the University of Illinois. Stored in liquid nitrogen |
| TrypLE | Life Technologies | 12605036 | Stored in 100mL at room temperature |
| Dulbecco’s modified Eagle medium | Cell Media Facility at School of Chemical Sciences at UIUC | 50003PC | Supplier: Corning |
| Nonessential amino acids | Cell Media Facility at School of Chemical Sciences at UIUC | 25-025-CI | Already added into DMEM by facility. Supplier: Corning |
| 10% fetal bovine serum | Fisher Scientific | 03-600-511 | Stored in 500mL at < -10⁰C |
| 1% Penicillin–Streptomycin | Life Sciences Storeroom at UIUC | 17602e | Supplier: VWR Stored in 100 ml at 4⁰C |
| Cell scraper | Fisher Scientific | 12-565-58 | Small 23cm 50 pack |
| Cell Dissociation Solution | Corning | MT-25-056CI | Used to lift cells non-enzymatically for the use in cell experiments |
| Hemacytometer | Hausser | 02-671-54 | Used to count cells for quantification of cell solutions and capture and release effectivity. |
| Biotin | Amresco | 58-85-5 | Used to release cells from surface. |
| HBSS | Created from Recipe | N/A | Used to keep cells alive in suspension as well as wash surfaces of non-specific binding. (Adapted from Cold Spring Harbor Protocols): In 500 mL, use 4 g NaCl, .2 g KCl, .0402 g Na2PO4*7H2O, .03 g KH2PO4 and .5 g Glucose. Add DI water to get to 500 mL, filter, and then refrigerate. |
| HLA-ABC Antibody | BioLegend | 311402 | Antibody used to capture MCF7gfp cells |
| hIgG Antibody | BioLegend | HP6017 | Antibody used to capture MCF7gfp cells |
| MCF7 GFP cells | Cell Biolabs | AKR-211 | Luminal Breast Cancer line that has been transfected with green fluorescent protein. |
| Assorted Conicals | Thermo-Scientific | 15mL: 12-565-268 | 50/15 mL plastic conicals for storing solutions and aliquots. |
| Mini-Tube Rotators (End over End Mixer) | Fisher Scientific | 05-450-127 | Used to incubate antibody and mix other cellular solutions in order to mix |
| Axiovert 200M (Fluorescence Microscope) | Zeiss | N/A | Zeiss Axiovert 200 M inverted florescence microscope. |
| Zeba Desalting columns | Thermo-Scientific | PI-87770 | Used to purify newly biotinylated antibodies after the use of the Biotinylation Kit. Instructions provided at: http://www.funakoshi.co.jp/data/datasheet/PCC/89894.pdf |
| EZ Link Sulfo NHS Low Weight Biotinylation Kit | Thermo- Scientific | Used to biotinylate antibodies to allow them to integrate with the capture surface | |
| Plate Reader | BioTek | Synergy HTX Multimode Reader | Used to quantitatively measure fluorescent intensity in the titration experiments. |
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