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Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
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Neuroscience

Kortsiktige fritt flytende Slice kulturer fra Adult Human Brain

Published: November 05, 2019
doi:
10.3791/59845
, , , , , , , , ,
1Department of Biochemistry and Immunology, Ribeirão Preto Medical School,University of São Paulo, 2Department of Physiology, Ribeirão Preto Medical School,University of São Paulo, 3Department of Pathology and Forensic Medicine, Ribeirão Preto Medical School,University of São Paulo, 4Department of Anatomy, Institute of Biosciences,São Paulo State University, 5Clinical Hospital at the Ribeirão Preto Medical School,University of São Paulo

Summary

En protokoll for å forberede fritt flytende skive kulturer fra voksen menneskelige hjerne er presentert. Protokollen er en variant av den mye brukte Slice kultur metoden ved hjelp av membran inserts. Det er enkelt, kostnadseffektivt, og anbefales for å kjøre kortsiktige analyser som tar sikte på å løse mekanismer for neurodegeneration bak alder-tilknyttede hjernesykdommer.

Abstract

Organotypic, eller Slice kulturer, har vært mye brukt til å modellere aspekter av sentralnervesystemet fungerer in vitro. Til tross for potensialet i skive kulturer i nevrovitenskap, studier ved hjelp av voksen nervøs vev for å forberede slike kulturer er fortsatt knappe, spesielt de fra menneskelige. Bruk av voksen menneskelig vev for å forberede Slice kulturer er spesielt attraktivt å forsterke forståelsen av menneskelig neuropathologies, som de holder unike egenskaper typisk for den modne menneskelige hjerne mangler i skiver produsert fra gnager (vanligvis nyfødt) nervøs vev. Denne protokollen beskriver hvordan du bruker hjernevevet samlet inn fra levende menneskelige donorer sendt til resective hjernen kirurgi for å forberede kortsiktige, fritt flytende skive kulturer. Prosedyrer for å opprettholde og utføre biokjemiske og cellebiologi analyser ved hjelp av disse kulturene er også presentert. Representative resultater viser at den typiske menneskelige kortikale laminering er bevart i skiver etter 4 dager in vitro (DIV4), med forventet tilstedeværelse av de viktigste nevrale celletyper. Videre skiver på DIV4 gjennomgår robust celle død når utfordret med en giftig stimulans (H2O2), noe som indikerer potensialet i denne modellen til å tjene som en plattform i celle død analyser. Denne metoden, et enklere og kostnadseffektivt alternativ til den mye brukte protokollen ved hjelp av membran innsatser, er hovedsakelig anbefalt for å kjøre kortsiktige analyser som tar sikte på å løse mekanismer for neurodegeneration bak alder-tilknyttede hjernesykdommer. Til slutt, selv om protokollen er viet til å bruke kortikale vev samlet inn fra pasienter som sendes til kirurgisk behandling av pharmacoresistant Temporal flik epilepsi, hevdes det at vev samlet inn fra andre hjerneregioner/forhold bør også være betraktes som kilder til å produsere lignende fritt flytende skive kulturer.

Introduction

Bruken av menneskelige prøver i forskning er utvetydig en flott mulighet til å studere menneskelige hjerne patologi, og moderne teknikker har åpnet nye måter for robust og etisk eksperimentering med pasient-avledet vev. Metoder som organotypic/Slice kulturer tilberedt fra voksne menneskelige hjerne har blitt stadig mer brukt i paradigmer som optogenetics1, elektrofysiologi2,3,4,5, plastisitet 6,7,8,9, nevrotoksisitet/neuroprotection10,11,12,13, Cell Therapy14, Drug screening15,16,17, genetikk og genredigering12,18,19,20, blant andre, som en strategi for bedre forstå nevrologiske sykdommer i voksen alder.

Forståelsen av mekanismer underliggende menneskelige hjerne patologi avhenger eksperimentelle strategier som krever et stort antall. Omvendt, i tilfelle av Slice kulturer, selv om tilgang til menneskelige prøver er fortsatt vanskelig, muligheten for å generere opptil 50 skiver fra en enkelt kortikale prøve delvis omgår kravet om å rekruttere flere frivillige ved å øke antall replikerer og utførte analyser per innsamlet vev21.

Flere protokoller for Brain organotypic/Slice kulturer har blitt beskrevet, alt fra den klassiske oculo utkast22,23 til roller tube24,25,26, halvgjennomtrengelig membraner Interface27,28,29,30, og fritt flytende skiver31,32. Avhengig av særtrekk av en eksperimentell design, har hver teknikk sine egne fordeler og ulemper. Kortsiktige, fritt flytende skiver kulturer fra voksne menneskelige hjerner er i noen tilfeller fordelaktig over metoden som brukes av Stoppini et al.27, hvis vurderer det faktum at selv om langsiktig celle overlevelse in vitro er vanligvis et stort problem når du vurderer en kultur metode, i mange eksperimenter bare korte perioder i kulturen er nødvendig12,31,32,33,34,35. Under disse forholdene, presenterer bruk av frittflytende kulturer fordelen av å være enklere og mer kostnadseffektivt, samt mer nøyaktig likner den opprinnelige menneskelige vev tilstand enn skiver holdt i kultur over 2-3 uker.

Til tross for potensialet i Slice kulturer til nevrovitenskap, studier ved hjelp av voksen nervøs vev for å forberede slike kulturer er fortsatt knappe, spesielt fra menneskelige. Denne artikkelen beskriver en protokoll for å bruke samlet hjernevev fra levende menneskelige donorer sendt til resective hjernen kirurgi for å forberede fritt flytende skive kulturer. Prosedyrer for å opprettholde og utføre biokjemiske og cellebiologi analyser ved hjelp av disse kulturene er detaljert. Denne protokollen har vist seg verdifullt for å analysere levedyktighet og neuronal funksjon i undersøkelser på mekanismer av neuropathologies knyttet til voksenlivet.

Protocol

Live voksen hjernevev ble innhentet fra pasienter som gjennomgår resective nevrokirurgi for behandling av pharmacoresistant Temporal flik epilepsi (figur 1A). Alle prosedyrer ble godkjent av etikk komiteen fra klinikker Hospital ved Ribeirão Preto Medical School (17578/2015), og pasienter (eller deres juridiske ansvarlig person) avtalt og signerte informerte samtykke vilkår. Innsamling av vevet ble gjort av nevrokirurgi teamet ved epilepsi Surgery Center (CIREP-klinikker …

Representative Results

Et kritisk aspekt for å evaluere kvaliteten og helsen til kulturperler skiver er tilstedeværelsen og typisk morfologi av de forventede nevrale celletyper, neurons, og gliacellene celler. Den typiske arkitekturen av den menneskelige kortikale laminering ble observert i en skive på DIV4, avslørt av neuronal immunolabeling (figur 2D). I tillegg ble det også observert forventet tilstedeværelse av mikroglia og astroglia (figur 2B, C</st…

Discussion

Denne protokollen for å produsere fritt flytende, kortsiktige Slice kulturer er en alternativ metode for dyrking voksne menneskelige neocortical skiver. En slik protokoll for Slice kulturer kan være mottagelig for studier på (men ikke begrenset til) optogenetics1,44,45, elektrofysiologi2,3,4,5 , kor…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbeidet er støttet av FAPESP (Grant 25681-3/2017 til AS), KAPPER (post-doktor fellesskap PNPD/INCT-HSM til A.F. og pre-doktor fellesskap til N.D.M.) og FAEPA. G.M.A. har en Master ‘ s fellesskap fra FAPESP (MS 2018/06614-4). N.G.C. har en CNPq Research Fellowship. Vi takker pasientene og deres familier for å donere den resected vev for denne studien. Vi vil gjerne erkjenne støtte fra beboere, sykepleiere, teknikere, og CIREP team, fra Clinical Hospital ved Ribeirão Preto Medical School, Universitetet i São Paulo, som hjalp i ulike stadier av prosessen.

Materials

2-Propanol Merck 1096341000
Acrylamide/Bis-Acrylamide, 30% solution Sigma Aldrich A3449 
Agarose Sigma Aldrich A9539
Ammonium persulfate Sigma A3678-25G
Amphotericin B Gibco 15290-018
Antibody anti-ERK 2 (rabbit) Santa Cruz Biotecnology sc-154 Dilution 1:1,000 in BSA 2.5%
Antibody anti-pERK (mouse) Santa Cruz Biotecnology sc-7383 Dilution 1:1,000 in BSA 2.5%
B27 Gibco 17504-044
BDNF Sigma Aldrich SRP3014
Bovine Serum Albumin Sigma Aldrich A7906
Bradford 1x Dye Reagent BioRad 500-0205
EDTA Sigma T3924 Used in RIPA buffer
Glucose Merck 108337
Glutamax Gibco 35050-061
Hank's Balanced Salts Sigma Aldrich H1387-10X1L
Hepes Sigma Aldrich H4034
Hydrochloric acid Merck 1003171000
Hydrogen Peroxide (H2O2) Vetec 194
Mouse IgG, HRP-linked whole Ab (anti-mouse) GE NA931-1ML
NaCl Merck 1064041000 Used in RIPA buffer
Neurobasal A Gibco 10888-022
Non-fat dry milk (Molico) Nestlé Used for membrane blocking
PBS Buffer pH 7,2 Laborclin 590338
Penicilin/Streptomicin Sigma Aldrich P4333
Potassium Chloride Merck 1049361000
Prime Western Blotting Detection Reagent GE RPN2232
Rabbit IgG, HRP-linked whole Ab (anti-rabbit) GE NA934-1ML
SDS Sigma L5750 Used in RIPA buffer
TEMED GE 17-1312-01
Thiazolyl Blue Tetrazolium Bromide (MTT) Sigma Aldrich M5655
Tris Sigma T-1378 Used in RIPA buffer
Triton x-100 Sigma X100 Used in RIPA buffer
Ultrapure Water Millipore Sterile water, derived from MiliQ water purification system
Equipment and Material
24-well plates Corning CL S3526 Flat Bottom with Lid
Amersham Potran Premium (nitrocellulose membrane)  GE 29047575
Carbogen Mixture White Martins 95% O2, 5% CO2
CO2 incubator New Brunswick Scientific CO-24 Incubation of slices 5% CO2, 36ºC
Microplate Reader Molecular Devices
Microtubes Greiner 001608 1,5mL microtube
Motorized pestle Kimble Chase
Plastic spoon Size of a dessert spoon
Razor Blade Bic Chrome Platinum, used in slicing with vibratome
Scalpel Blade Becton Dickinson (BD) Number 24 Used for slicing of tissue; recommended same size or smaller
Superglue (Loctite Super Bonder) Henkel Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6'-di-terc-butil-2,2'-metilenodi-p-cresol; homopolymer
Vibratome  Leica 14047235612 – VT1000S
Name of Material/ Equipment for Immunohistochemistry
Antibody anti-NeuN (mouse) Millipore  MAB377 Dilution 1:1,000 in Phosphate Buffer
Antibody anti-GFAP (mouse) Merck MAB360 Dilution 1:1,000 in Phosphate Buffer
Antibody anti-Iba1 (rabbit) Abcam EPR16588 – ab178846 Dilution 1:2,000 in Phosphate Buffer
Biotinylated anti-mouse IgG Antibody (H+L) Vector BA-9200
DAB Sigma Aldrich D-9015
Entellan Merck 107960
Ethanol Merck 1.00983.1000
Gelatin Synth 00G1002.02.AE Used for coating slides
Microtome Leica SM2010R Equipped with Freezing Stage (BFS-10MP, Physiotemp), set to -40ºC
Normal Donkey Serum Jackson Immuno Research 017-000-121
Paraformaldehyde Sigma Aldrich 158127
Rabbit IgG, HRP-linked whole Ab (anti-rabbit) GE NA934-1ML
Slides (Star Frost) Knittel Glaser Gelatin coated slides
Sucrose Vetec 60REAVET017050
Vectastain ABC HRP Kit (Peroxidase, Standard) Vector PK-4000, Kit Standard
Xylene Synth 01X1001.01.BJ
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Brain Slice CultureFree-floating Slice CultureBrain SurgeryNeurodegenerationCell BiologyImmunohistochemistryVibratomeAgaroseSlicingTransportCarbogen

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Fernandes, A., Mendes, N. D., Almeida, G. M., Nogueira, G. O., Machado, C. d. M., Horta-Junior, J. d. A. d. C., Assirati Junior, J. A., Garcia-Cairasco, N., Neder, L., Sebollela, A. Short-Term Free-Floating Slice Cultures from the Adult Human Brain. J. Vis. Exp. (153), e59845, doi:10.3791/59845 (2019).
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