Un metodo di isolamento delle cellule mirata tramite Glass funzionalizzazione superficiale
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
Da banco approcci separazione cella corrente (ad esempio, la fluorescenza delle cellule attivate di smistamento 1, cattura laser micro-dissezione 2, immuno-magnetico tallone di separazione 1) può richiedere diverse ore di preparazione e smistamento. Questi grandi scale temporali possono influenzare i livelli di risposta e di espressione fisiologici, con conseguente analisi che non sono rappresentativi della risposta fisiologica 3. I sistemi sono necessari che possono rapidamente ed efficacemente isolare specifici tipi cellulari senza interrompere recettore livelli di superficie cellulare per migliorare l'isolamento delle cellule e arricchimento per applicazioni biomediche. Pertanto, il razionale per il nostro approccio è quello di sviluppare un approccio dolce per l'isolamento delle cellule.
Il "lab on a chip" concetto offre la promessa di ordini di grandezza più veloce di isolamento cellulare (ore-to-minuti), e coinvolge più frequentemente catturare cellule su una superficie e rilasciare cellule o conte intracellularenti attraverso fisico 4,5 o metodi chimici 6. Anche se questi approcci offrono alcuni vantaggi, quali l'identificazione dell'espressione proteica 7,8, individuando espressione di RNA 9-11, o addirittura fornire cellule per coltura in vitro 12,13, molte di queste tecniche non può essere tradotto per la diagnostica, come recettore delle cellule profilatura a causa ai loro ambienti non fisiologici. Agenti di sollevamento enzimatici come la collagenasi può colpire anche queste quantità del recettore 14,15, cioè le tecniche di quantificazione recettore delle cellule che utilizzano questi agenti di sollevamento non genererà dati fisiologici precisi. Lisi cellulare impedisce differenziazione tra i recettori di superficie nativi e quelli precedentemente internalizzato 16. Questo protocollo descrive un approccio rapido e delicato per l'isolamento delle cellule.
Protocol
Representative Results
Discussion
Miglioramenti nelle tecniche di isolamento delle cellule promuove studi scientifici nelle relazioni struttura-funzione in neuroscienze 18, staminali programmazione delle cellule in biologia rigenerativa, e la segnalazione angiogenico in biologia vascolare 19. Infatti, colture primarie 20 (ad esempio, HUVEC) in biologia vascolare avviene principalmente attraverso l'uso di tecniche di isolamento cellulare. Isolamento delle cellule è stata recentemente utilizzata anche per il …
Divulgazioni
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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