JoVE Journal > Neuroscience
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Neuroscience

Kortsigtede fritflydende udsnit kulturer fra den voksne menneskelige hjerne

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 protokol til at forberede fritflydende skive kulturer fra voksne menneskelige hjerne er præsenteret. Protokollen er en variation af den udbredte skive kultur metode ved hjælp af membran skær. Det er simpelt, omkostningseffektivt, og anbefales til at kørekort sigtede assays sigter mod at opklare mekanismer af neurodegeneration bag aldersrelaterede hjernesygdomme.

Abstract

Organotypiske, eller skive kulturer, er blevet bredt anvendt til at modellere aspekter af det centrale nervesystem fungerer in vitro. På trods af potentialet i Skive kulturer i neurovidenskab, undersøgelser ved hjælp af voksne nervevæv til at forberede sådanne kulturer er stadig knappe, især dem fra menneskelige. Brugen af voksne menneskelige væv til at forberede skive kulturer er særligt attraktivt at forbedre forståelsen af humane neuropatiologier, da de har unikke egenskaber typisk for den modne menneskelige hjerne mangler i skiver fremstillet af gnaver (normalt neonatal) nervevæv. Denne protokol beskriver, hvordan man bruger hjernevæv indsamlet fra levende menneskelige donorer indsendt til resective hjernekirurgi til at forberede kortsigtede, fritflydende skive kulturer. Procedurer til at vedligeholde og udføre biokemiske og cellebiologi assays ved hjælp af disse kulturer er også præsenteret. Repræsentative resultater viser, at den typiske humane kortikale laminering bevares i skiver efter 4 dage in vitro (DIV4), med forventet tilstedeværelse af de vigtigste neurale celletyper. Desuden, skiver på DIV4 undergår robust celledød, når udfordret med en giftig stimulus (H2O2), hvilket indikerer potentialet i denne model til at fungere som en platform i celledød assays. Denne metode, et enklere og omkostningseffektivt alternativ til den udbredte protokol ved hjælp af membran skær, anbefales primært til at kørekort varige assays, der har til formål at opklare mekanismer af neurodegeneration bag aldersrelaterede hjernesygdomme. Endelig, selv om protokollen er afsat til anvendelse af kortikale væv indsamlet fra patienter indsendt til kirurgisk behandling af farmakoresistant temporale lobe epilepsi, det hævdes, at væv indsamlet fra andre hjerneregioner/betingelser bør også være betragtes som kilder til at producere lignende fritflydende udsnit kulturer.

Introduction

Brugen af humane prøver i forskning er utvetydigt en stor mulighed for at studere menneskelige hjerne patologier, og moderne teknikker har åbnet nye måder for robuste og etiske eksperimenter ved hjælp af patient-afledte væv. Metoder som organotypic/skive kulturer fremstillet af voksne menneskelige hjerne er blevet mere og mere anvendt i paradigmer såsom optogenetik1, Elektrofysiologi2,3,4,5, plasticitet 6,7,8,9, neurotoksicitet/neuroprotection10,11,12,13, celleterapi14, Drug screening15,16,17, genetik og genredigering12,18,19,20, blandt andre, som en strategi for bedre at forstå neurologiske sygdomme i voksenalderen.

Forståelsen af mekanismer underliggende menneskelige hjerne patologier afhænger af eksperimentelle strategier, der kræver et stort antal emner. Omvendt, i tilfælde af skive kulturer, selv om adgang til humane prøver stadig er vanskelig, muligheden for at generere op til 50 skiver fra en enkelt kortikal prøve delvist omgår kravet om rekruttering af flere frivillige ved at øge antal replikater og udførte assays pr. indsamlet væv21.

Flere protokoller for hjernen organotypic/skive kulturer er blevet beskrevet, spænder fra den klassiske oculo udkast22,23 til rullerør24,25,26, semi-permeable membraner grænseflade27,28,29,30og fritflydende udsnit31,32. Afhængigt af de særlige forhold i et eksperimentelt design, hver teknik har sine egne fordele og ulemper. Kortvarige, fritflydende skiver kulturer fra voksne menneskelige hjerner er i nogle tilfælde fordelagtige over den metode, der anvendes af Stoppini et al.27, hvis man tager i betragtning, at selv om langvarig celle overlevelse in vitro normalt er et stort problem, når man evaluerer en kultur metode, i mange eksperimenter kun korte perioder af tid i kulturen er nødvendige12,31,32,33,34,35. Under disse betingelser, brugen af fritflydende kulturer præsenterer fordelen ved at være enklere og mere omkostningseffektiv, samt mere præcist ligner den oprindelige menneskelige væv tilstand end skiver holdes i kulturen over 2-3 uger.

På trods af potentialet i Skive kulturer til neurovidenskab, er undersøgelser ved hjælp af voksne nervevæv til at forberede sådanne kulturer stadig knappe, især fra menneskelige. Denne artikel beskriver en protokol til at bruge indsamlede hjernevæv fra levende menneskelige donorer indsendt til resective hjernekirurgi til at forberede fritflydende skive kulturer. Procedurer til at vedligeholde og udføre biokemiske og cellebiologi assays ved hjælp af disse kulturer er detaljerede. Denne protokol er blevet bevist værdifuld for at analysere levedygtighed og neuronal funktion i undersøgelser af mekanismerne i neuropatiologier knyttet til voksenalderen.

Protocol

Levende voksne hjernevæv blev opnået fra patienter, der gennemgik reektiv Neurokirurgi til behandling af farmakoresistant temporale lobe epilepsi (figur 1A). Alle procedurer blev godkendt af den etiske komité fra klinikker Hospital på Ribeirão Preto Medical School (17578/2015), og patienter (eller deres juridiske ansvarlige person) aftalt og underskrevet informeret samtykke vilkår. Indsamlingen af vævet blev udført af neuro kirurgi team på epilepsi kirurgi Center (C…

Representative Results

Et kritisk aspekt for at evaluere kvaliteten og sundheden af dyrkede skiver er tilstedeværelsen og typiske morfologi af de forventede neurale celletyper, neuroner, og gliaceller celler. Den typiske arkitektur af den menneskelige kortikale laminering blev observeret i en skive på DIV4, afsløret af neuronal immunolabeling (figur 2D). Desuden blev den forventede tilstedeværelse af microglia og astroglia (figur 2B, C) også obse…

Discussion

Denne protokol til produktion af fritflydende, kortvarige skive kulturer er en alternativ metode til dyrkning af voksne menneskelige neortikale skiver. En sådan protokol for skive kulturer kan være modtagelig for undersøgelser af (men ikke begrænset til) optogenetik1,44,45, Elektrofysiologi2,3,4,5 …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde understøttes af FAPESP (Grant 25681-3/2017 to AS), CAPES (post-doktor Fellowship PNPD/INCT-HSM til A.F. og præ-doktor stipendiat til N.D.M.) og FAEPA. G.M.A. har en Master’s Fellowship fra FAPESP (MS 2018/06614-4). N.G.C. besidder et CNPq Research Fellowship. Vi takker patienterne og deres familier for at donere det viderekomne væv til denne undersøgelse. Vi vil gerne anerkende støtte fra beboere, sygeplejersker, teknikere og CIREP team, fra det kliniske Hospital på Ribeirão Preto Medical School, University of São Paulo, der hjalp i forskellige stadier af processen.

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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Tags

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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