Une méthode d'isolement cellulaire ciblée via verre fonctionnalisation de surface
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
Paillasse Les approches actuelles de séparation cellulaire (par exemple, cellules activées par fluorescence de tri 1, capture laser micro-dissection 2, immuno-bille magnétique séparation 1) peut prendre plusieurs heures de préparation et de tri. Ces grandes échelles de temps peuvent influer sur les niveaux d'intervention et d' expression physiologiques, résultant dans les analyses qui ne sont pas représentatifs de la réponse physiologique 3. Les systèmes sont nécessaires qui peuvent rapidement et efficacement isoler des types cellulaires spécifiques sans perturber le récepteur des niveaux de surface cellulaire afin d'améliorer l'isolement cellulaire et d'enrichissement pour des applications biomédicales. Par conséquent, la justification de notre approche est de développer une approche douce pour l'isolement cellulaire.
Le "laboratoire sur puce" concept offre la promesse d'ordres de grandeur plus rapide (heures à minutes) l'isolement cellulaire, et implique le plus souvent la capture des cellules sur une surface et de libérer des cellules ou intracellulaire contents par le biais physique 4,5 ou des méthodes chimiques 6. Bien que ces approches offrent quelques avantages tels que l' identification expression de protéines de 7,8, en identifiant l' expression de l' ARN 9-11, ou même fournir des cellules de culture in vitro 12,13, bon nombre de ces techniques ne peuvent pas être traduits à des diagnostics tels que le profilage de récepteur des cellules due à leurs environnements non-physiologiques. Agents de levage Enzymatic tels que les collagénases peuvent également affecter ces quantités de récepteurs 14,15, ce qui signifie des techniques de quantification du récepteur des cellules qui utilisent ces agents de levage ne génère pas des données physiologiques précises. La lyse cellulaire prévient la différenciation entre les récepteurs de la surface d' origine, et celles qui ont déjà été internalisée 16. Ce protocole décrit une approche rapide et douce pour l'isolement cellulaire.
Protocol
Representative Results
Discussion
L' amélioration des techniques d'isolement cellulaire favorise d' études scientifiques dans les relations structure-fonction en neurosciences 18, la tige de programmation de cellules en biologie régénérative, et la signalisation angiogénique en biologie vasculaire 19. En effet, la culture de cellules primaires 20 (par exemple, les HUVEC) dans la biologie vasculaire se fait principalement par l'utilisation de techniques d'isolement des cellules. L' i…
Disclosures
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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