Karakterisering af Humane monocytafledte dendritiske celler fra Imaging flowcytometri: en sammenligning mellem to Monocyt Isolation protokoller
Summary
This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.
Abstract
Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 – 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.
Introduction
Dendritiske celler (DCS) er væsentlige mediatorer af medfødte og adaptive immunforsvar. De fungerer til at fremkalde primære immunrespons og lette udviklingen af immunologisk hukommelse. Disse celler er primært ansvarlige for antigen capture, migration og T-celle-stimulering og betegnes derfor som professionelle antigenpræsenterende celler (APC'er) 1 .Manipulation af DC'er kunne bruges på tværs af en bred vifte af forskningsområder og i det kliniske miljø at behandle forskellige inflammatoriske sygdomme, såsom HIV 6,7, cancer 8, autoimmune sygdomme 9 og allergiske reaktioner 10. Udviklingslandene bliver også brugt til stofmisbrug forskning for at løse ukendte mekanismer og veje såsom dem forbundet med alkoholafhængighed 11-14, narkotikamisbrug 13,15, og kombinationen af HIV-infektion og stofmisbrug 16-19. Disse igangværende undersøgelser og fremtidige undersøgelser In området immunologi gør in vitro generation af DC'er ekstremt vigtigt for forskning. Der er imidlertid adskillige vanskeligheder forbundet med at isolere DC'er fra humant blod som de kun udgør 0,1 – 1% af totale mononukleære blodceller 20.
Til dato er nogle af de veletablerede metoder til frembringelse af DC'er in vitro består af plast eller glas adhærens af monocytter 21,22, densitetsgradientcentrifugering 23, specifik markør baseret separation, såsom magnetisk cellesortering 22, fluorescerende cellesortering 24, positiv selektion af CD14 + monocytter under anvendelse dextran-coatede magnetiske nanopartikler 25 og hurtig isolering af højtoprensede monocytter under anvendelse fuldautomatisk negativ celleselektion 26. Imidlertid er den bedste metode til valg forbliver kontroversiel. Derfor, for at forbedre DC generation teknikker, er adskillige fremgangsmåder blevet udviklet, hvorirenheden af disse celler kan forøges meget ved differentiering fra oprensede CD34 + stamceller og monocytter isoleret fra mononukleære celler fra perifert blod (PBMC'er) 27. Som nævnt før, en udbredt og populær metode til at generere monocytderiverede dendritiske celler (MDDCs) er at undersøge muligheden af monocytter til at holde sig til glas eller plast (vedhæftning metode) 21,22,27. Vedhæftningen metode er en hurtig og enkel metode, der ikke kræver anvendelse af kompliceret udstyr. Men nogle ulemper ved denne proces, omfatter lymfocyt kontaminering, lav fleksibilitet, og monocyt forbigående manipulation 28. En alternativ fremgangsmåde til overholdelse metode er den magnetiske isolation af monocytter fra total PBMC'er, især med anvendelse af en human monocyt berigelse kit, som er designet til at isolere monocytter fra PBMC'er ved negativ selektion 26. Under denne procedure, er uønskede celler målrettet til fjernelse med tetramertantistof-komplekser og dextran-coatede magnetiske partikler. Fordelen ved denne isolation fremgangsmåde er, at de uønskede mærkede celler separeres ved anvendelse af en magnet, mens målceller kan frit hældes ud i et nyt rør uden behov for kolonner. Til dato med tilgængeligheden af specifikke monoklonale antistoffer, der kan oprette etiketter unikke cellepopulationer, har den magnetiske separation teknik bliver ikke blot en yderligere metode, men en nødvendighed for isolering af sjældne celler i området immunologi. For eksempel teknikker såsom magnetisk cellesortering med kommercielt tilgængelige paramagnetiske MACS-nanopartikler har lettet udviklingen af nye metoder til forskning og kliniske anvendelser 22,29. Desuden har nyere videnskabelige undersøgelser, der sammenligner DC generation fra monocytadhærens og MACS teknologiske metoder viste en højere DC renhed og levedygtighed ved hjælp af MACS adskilte monocytter 22,30.
Den aktuelle undersøgelse gaveren sammenligning mellem to metoder til generering af humane DC'er fra monocytter isoleret fra PBMC'er: 1) monocyt isolering ved adhærens og 2) monocyt isolering ved negativ selektion under anvendelse af et kommercielt humant monocyt berigelse kit. Denne undersøgelse giver bevis for, at den negative selektion magnetiske separation procedure for at isolere monocytter genererer det højeste udbytte af monocytter med ingen signifikante forskelle i monocyt levedygtighed sammenlignet med monocytter isoleret ved vedhæftning metode. Til gengæld, efter syv dage, monocytterne isoleret ved magnetisk separation differentieret til MDDCs med signifikant højere proliferativ kapacitet og højere mængde celler, der udtrykker dobbelt positive (CD11c + / CD14 +) fænotype uden at påvirke MDDC levedygtighed. Samlet er den nuværende undersøgelse adskiller sig fra de tidligere undersøgelser refereres ovenfor, da det viser evnen hos begge teknikker til samtidig generere monocytter, der er i stand til at proliferere og differentiere til CD11c +MDDCs (> 70%) efter syv dage i kultur uden at kompromittere deres levedygtighed. Hertil kommer, at nuværende tilgang giver for første gang karakterisering af forskellige CD11c / CD14 MDDCs befolkninger ved billedbehandling flowcytometri.
Sammenfattende da DC'er spiller et knudepunkt rolle vedrørende forskning inden for immunologi, skal der tages forskellige parametre i betragtning, når man overvejer, hvordan de er beregnet, samt hvilke metoder der bruges til at isolere og kultur dem in vitro. Derfor er denne undersøgelse har til formål at give indsigt på to forskellige metoder til monocyt isolation, og hvordan disse metoder differentielt påvirke monocyt levedygtighed og udbytte i sidste ende påvirker dendritiske cellers levedygtighed, proliferation og fænotype. Disse resultater vil i høj grad bidrage til området for immunologi og vil give en detaljeret protokol af DC isolation, rensning og karakterisering.
Protocol
Representative Results
Discussion
Baseret på de kendte vanskeligheder isolere og generere MDDCs fra humant blod, nærværende undersøgelse havde til formål at give en samlet sammenligning af to veletablerede metoder til generering af MDDCs. Den første metode sammenlignes, er en velkendt metode til generering MDDCs ved at udnytte evnen hos monocytter at klæbe til glas eller plast (adhærens metode) 21,22,27. Vedhæftningen metode er hurtig og omkostningseffektiv, og kræver ikke anvendelse af komplekst udstyr. Men nogle ulemper ved denne …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research is supported by the National Institute on Alcohol Abuse and Alcoholism, award K99/R00 AA021264. Additional lab support as part of startup package has been received from the Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, and FIU- Office of Research and Economic Development. Gianna Casteleiro was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Materials
| Ficoll-Paque | GE Healthcare | 17-5442-03 | Must be used at room temperature |
| Phosphate-buffered saline (PBS) | Life Technologies | 10010-023 | |
| ACK Lysing buffer | Quality Biological | 118-156-101 | |
| RPMI 1640 medium | Life Technologies | 22400-089 | |
| Antibiotic-Antimycotic (100X) | Life Technologies | 15240-062 | |
| Fetal Bovine Serum (FBS) | Life Technologies | 16000-044 | |
| RoboSep buffer | StemCell | 20104 | |
| EasySep Human monocyte enrichment kit | StemCell | 19059 | |
| TC20 Automated cell counter | Bio-Rad | 145-0101 | |
| FITC-Dextran | Sigma Aldrich | FD4-100MG | |
| Trypan blue stain (0.4%) | Life Technologies | 15250-061 | |
| Synergy 2 multi-mode reader | Biotek | 7131000 | |
| XTT Sodium salt bioreagent (XTT) | Sigma Aldrich | X4626-100MG | |
| Dimethyl sulfoxide bioreagent (DMS) | Sigma Aldrich | D8418-500ML | |
| Thiazolyl blue tetrazolium bromide (MTT) | Sigma Aldrich | m5655-500MG | |
| Sodium Dodecyl Sulfate (SDS) | Bio-Rad | 161-0302 | |
| Phenazine Methosulfate (DMSO) | Sigma Aldrich | P9626-1G | |
| Inactivated (HI) Human Serum | Chemicon | S1-100ML | |
| Accuri C6 Flow Cytometer | BD Accuri | 653119 | |
| FlowSight Amnis Flow Cytometer | EMD Millipore | 100300 |
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