Summary
House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.
Abstract
HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 °C to stimulate cell proliferation and incubation at 39 °C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.
Introduction
House Ear Institute-Organ of Corti 1 (HEI-OC1) celler er afledt fra det auditive organ i en transgen mus 1,2. Inkubation af enhver celle fra denne transgene mus ved 33 ° C / 10% CO2 (tolerante betingelser) inducerer ekspression af et immortaliserende gen, der udløser de-differentiering og accelereret proliferation; flytte cellerne til 39 ° C / 5% CO 2 (ikke-tolerante betingelser) føre til nedsat proliferation, differentiering og, i det mindste i tilfælde af HEI-OC1, celledød 2,3.
HEI-OC1 celler blev klonet og karakteriseret i vores laboratorium over ti år siden, og de første undersøgelser viste, at de udtrykker specifikke markører for cochleare hårceller, såsom Prestin, myosin 7a, Atoh1, BDNF, calbindin og calmodulin, men også markører for støtte celler som connexin 26 og fibroblastvækstfaktorreceptor (FGF-R) 2. Derfor blev det foreslået, at HEI-OC1 kunne udgøre en common stamfader til sensoriske og understøttende celler i organ Corti 2. Parallelle undersøgelser forudsat stærke beviser for, at arketypiske ototoksiske stoffer som cisplatin, gentamicin og streptomycin induceret caspase-3 aktivering i disse celler, mens lægemidler anses ikke-ototoksisk, ligesom penicillin, ikke gjorde 2,3. Derfor blev denne cellelinje foreslået som et in vitro-system til at undersøge de cellulære og molekylære mekanismer involveret i ototoksicitet og til screening af potentielle ototoksicitet eller otoprotective egenskaber af nye farmakologiske stoffer. Det anslås, at HEI-OC1-celler er blevet anvendt i mere end et hundrede og halvtreds undersøgelser offentliggjort i de sidste ti år.
Ud fra følgende betragtninger ser på potentielle pro-apoptotiske effekt af forskellige stoffer var den største mål af de fleste af de undersøgelser med denne cellelinie, har andre vigtige celle processer som autofagi og ældning lige begyndt at blive undersøgt i HEI-OC1 celler 4-7. jegna nylig undersøgelse fra vores laboratorium 8, vi brugte HEI-OC1 celler til at indsamle et omfattende sæt af data om celledød, overlevelse, spredning, ældning og autofagi fremkaldt af forskellige farmakologiske stoffer, der ofte anvendes klinikken. Vi sammenlignede også nogle af svarene fra HEI-OC1 celler med dem fra HEK-293 (humane embryonale nyreceller) og HeLa (humane epitelceller) modtager samme behandling. Vores resultater indikerede, at HEI-OC1-celler reagerer på hvert lægemiddel i en karakteristisk måde, med en karakteristisk dosis- og tidsafhængig følsomhed over for mindst en af de mekanismer, der undersøges. Vi understregede også i denne undersøgelse, at en korrekt fortolkning af de eksperimentelle resultater vil kræve at udføre parallelle undersøgelser med mere end én teknik 8.
I en anden undersøgelse undersøgte vi brug af HEI-OC1-celler til at vurdere den funktionelle reaktion Prestin, motoren proteinet af cochleare ydre hårceller (OHCs) 9 </sop>. Vi rapporteret flowcytometri og konfokal laserscanningsmikroskopi undersøgelser på mønstret for Prestin udtryk, samt ikke-lineær kapacitans (NLC) og helcelle-patch fastspænding studier i HEI-OC1 celler dyrket på permissive (P-HEI-OC1) og ikke- tolerante (NP-HEI-OC1) betingelser. Vores resultater viste, at både total Prestin ekspression og plasmamembranen lokalisering stigning i en tidsafhængig måde i NP-HEI-OC1-celler. Interessant, vi fandt også, at stigningen i Prestin lokalisering på plasmamembran NP-HEI-OC1-celler korrelerede med en formindskelse i Na + K + ATPase, der omplantes fra plasmamembranen til cytoplasmaet uden væsentlige ændringer i den totale celleekspression. Derudover demonstrerede vi, at P-HEI-OC1 celler har en robust CNT tilknyttet Prestin motorisk funktion, der faldt når densiteten af Prestin molekyler til stede på plasmamembranen forøget. Tilsammen understøtter disse resultater kraftigt anvendeligheden af HEI-OC1-celler for at undersøge auditive proteiner.
I denne video artiklen beskriver vi, hvordan kultur HEI-OC1-celler, hvorfor det er hensigtsmæssigt at anvende celler vokser med permissive betingelser (P-HEI-OC1) for cytotoksicitet undersøgelser, hvordan man vurderer mekanismen / s af lægemiddel-induceret cytotoksicitet og hvordan at udføre elektrofysiologiske undersøgelser (f.eks, patch-clamp, ikke-lineær kapacitans (NLC)) for at undersøge funktionelle egenskaber Prestin, den molekylære motor af cochleære OHCs.
Protocol
Representative Results
Discussion
I denne rapport beskriver vi, hvordan kultur HEI-OC1 celler og bruge dem til at evaluere mekanismer lægemiddelinduceret cytotoksicitet og undersøge funktionelle egenskaber Prestin, den molekylære motor af cochlear OHCs. De tekniske procedurer er imidlertid generelt nok til nemt at kunne tilpasses til forskellige undersøgelser.
Alle her beskrevne protokoller kræver den korrekte brug af veletablerede cellekulturteknikker 10. Ligesom med enhver anden cellelinje, der arbejder med…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH Grants R01-DC010146 and R01-DC010397. Its content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.
Materials
| HEI-OC1 cells | ALL THE ASSAY KITS, EQUIPMENTS | ||
| Class II Biological Safety cabinet | The Baker Company | Sterilgard III | AND COMPANIES INDICATED IN THE |
| Refrigerated centrifuge | Eppendorf | 5810R | PREVIOUS 2 COLUMNS ARE ONLY |
| Inverted microscope | Zeiss | Axiovert 25 | EXAMPLES, AND ANY OTHER SIMILAR |
| Waterbath | Stovall | HWB115 | PRODUCT COULD BE USED. |
| Cell counter | Nexcelom | Cellometer Auto T4 | |
| Two (2) Cell incubators, one at 33°C/10% CO2 and other at 39°C/5% CO2 | Forma Scientific | 3110 | |
| Cell culture dishes, PS, 100 x 20 mm with vents | Greinier Bio-One | 664-160 | |
| Cell culture dishes , PS, 60 x 15 mm with vents | Greiner Bio-One | 628160 | |
| Cellstar tissue culture flasks 250 mL | Greiner Bio-One | 658-175 | |
| Cellstar tissue cultur flasks 550 mL | Greiner Bio-One | 660-175 | |
| 6 well cell culture plate, with lid-Cellstar | Greiner Bio-One | 657-160 | |
| Microtest Tissue culture plate, 96 well,flat bottom with lid | Becton Dickinson | 353072 | |
| Micro-Assay-Plate, Chimmey, 96-well white,clear botton | Greiner Bio-One | 655098 | |
| 50 ml Polypropylene conical tube with cap Cellstars | Becton Dickinson | 352070 | |
| 15 ml Polypropylene conical tubes with cap-Cellstars | Greiner Bio-One | 188-271 | |
| PBS pH 7.4 (1X) | Life Technologies | 10010-023 | |
| Dulbecco’s Modified Eagle’s Medium (DMEM) | Life Technologies | 11965-084 | |
| Fetal bovine serum (FBS) | Hyclone | SH10073.1 | |
| Leibovitz's L-15 Medium, no phenol red | Gibco/Invitrogen | 21083-027 | |
| Trypsin, 0.25% | Life Technologies | 25200-056 | |
| TACS MTT Cell Proliferation Assay Kit | Trevigen | 4890-25-K | |
| Caspase-Glo 3/7 Assay kit | Promega | G8091 | |
| BrdU Cell Proliferation Assay Kit | Cell Signaling | 6813 | |
| Non-enzymatic cell dissociation solution | Sigma-Aldrich | C5789 | |
| Cell-Tox Green Cytotoxicity Assay Kit | Promega | G8741 | |
| FACSAriaIII instrument | BD Biosciences | FACSAriaIII | With 488 nm excitation (blue laser) |
| Digital Blot Scanner | LI-COR | C-DiGit | |
| Electrophoresis and Blotting Unit | Hoefer | SE300 miniVE | |
| Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software | Molecular Devices | SpectraMax 5 | |
| Patch-clamp amplifier | HEKA | EPC-10 | |
| Puller for preparing patch electrodes | Sutter Instruments | P-97 |
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