Summary

En cGMP-gældende Expansion Metode til aggregater af humane neurale Stem og stamceller arvet fra pluripotente stamceller eller fostrets hjerne Tissue

Published: June 15, 2014
doi:

Summary

Denne protokol beskriver en ny mekanisk sønderdeling, der tillader udvidelse af sfæriske neurale stamceller og stamcelle aggregater uden dissociation til en enkelt cellesuspension. Fastholdelse af celle / celle kontakt giver mulighed for hurtig og stabil vækst i over 40 passager.

Abstract

En celle ekspansion teknik til at samle et stort antal celler fra et enkelt eksemplar til forskningsprojekter eksperimenter og kliniske forsøg vil være til stor gavn stamceller samfund. Mange nuværende ekspansion metoder er besværlige og dyre, og dem, der involverer komplet dissociation kan forårsage flere stamceller og stamceller typer til at gennemgå differentiering eller tidlig ældning. For at overvinde disse problemer har vi udviklet en automatiseret mekanisk passage benævnt "hakke", der er enkel og billig metode. Denne teknik undgår kemisk eller enzymatisk dissociation i enkelte celler og i stedet giver mulighed for storstilet udvidelse af suspenderet, sfæroidpartikler kulturer, der opretholder konstant celle / celle kontakt. Snittemetoden er primært blevet anvendt til fostrets hjerne-afledte neurale stamceller eller neurosfærer, og er for nylig blevet udgivet til brug med neurale stamceller fra embryoner og induceret pluripotente stamceller. Proceduren beliges seeding neurosfærer på en vævskultur petriskål og efterfølgende passerer en skarp, steril kniv gennem cellerne effektivt automatisere kedelige processen med manuelt mekanisk adskille hver sfære. Suspension af celler i kultur giver et gunstigt overfladeareal-til-volumen-forhold; som over 500.000 celler kan dyrkes i en enkelt neurosfære på mindre end 0,5 mm i diameter. I en T175 kolbe, kan over 50 millioner celler vokser i suspensionskulturer i forhold til kun 15 millioner i vedhængende kulturer. Vigtigere er det, snitning procedure blevet brugt i henhold til gældende god fremstillingspraksis (cGMP), tillader masse mængde produktion af klinisk-grade celle produkter.

Introduction

Der er en lang tradition for at udvide gnaver neurale stamceller i kultur som enten et monolag 1-3 eller aggregerede neurosfærer 4-7. Desuden har humane neurale stamceller (hNPCs) isoleret fra forskellige regioner i udviklingslandene centralnervesystemet 8-17 blevet udvidet in vitro. Disse celler er bi-potent, stand til at differentiere sig til både astrocytter og neuroner og har været et meget nyttigt værktøj i at studere neurale udvikling 18,19 og sygdom mekanisme 20,21. hNPCs er også blevet transplanteret ind i mange forskellige dyremodeller for sygdom i centralnervesystemet med varierende niveauer af integration, overlevelse og funktionelle virkninger 22-24.

Traditionelt er gnaver eller humane føtale afledt NPC udsættes for vækstfaktorer – ofte epidermal vækstfaktor (EGF) og / eller fibroblastvækstfaktor-2 (FGF-2) 25-28 – og både vedhæftende 29 og tre-Dimensionelle kugleformede systemer er typisk passage ved hjælp af enzymatisk dissociation i en enkelt-cellesuspension 30-34. Standarden metode til at udvide celler til forskning eller klinisk anvendelse er som tilhænger monolag på grund af let manipulation. Men vi har vist, at passage monolags og neurosfæreceller hNPCs med enzymatiske eller kemiske opløsninger resulterede i tidlig ældning 35.. Desuden kan enzymatisk dissociation resultere i forhøjede niveauer af differentiering og karyotypic abnormiteter baseret på data demonstreret med embryonale stamceller 36-38. Selv om standarden metode passaging hNPCs har produceret god fremstillingspraksis (cGMP) grade produkter, der er gået ind i fase 1 kliniske forsøg (Stamceller Inc., Neuralstem Inc.), den metode kun tilladt et par runder celleamplifikation, hvilket begrænser bank potentiale.

Det er klart, kan store forskningscentre eksperimenter og fremtidige kliniske forsøg drage fordel af muligheden for atudbrede celler i bulk og med forsinket senescens at tillade storstilet vækst og celle bankvirksomhed. For at imødekomme dette behov, har vi udviklet en ny og automatiseret måde mekanisk passaging intakte neurosfærer med "hakke" dem ind i små klynger for at opretholde celle-til-celle kontakt. Denne fremgangsmåde i høj grad øget deres levetid 39 og suspensionskultur tillader en mere effektiv udnyttelse af inkubator plads i forhold til monolag kulturer, som det ses med et alternativt 3D bioreaktor dyrkningsmetode 40. Den angivne hakke-protokollen giver mulighed for produktion af store banker fra en føtal prøve større end passage 10, en usandsynlig bedrift ved hjælp af standard passaging metoder. Selv om denne fremgangsmåde for passage hNPCs er utraditionel, det er stigende i popularitet, og blev for nylig offentliggjort med andre celletyper, såsom neurale stamceller fra humane embryonale og inducerede pluripotente stamceller, der gør det muligt i stor skala udvidelse til forskellige applikationer herunder vitro sygdom modellering 41-46. Vigtigere er det, har en cGMP-grade hNPC celle banken allerede er produceret med Snittemetoden, viser, at teknikken kan anvendes over for fremtidige kliniske anvendelser.

Protocol

1.. Etisk erklæring og sikkerhed Denne fremgangsmåde involverer brugen af ​​cellekultur produkter fra mennesker eller dyr. Alle afledte væv skal godkendes før brug af den relevante Institutional Review Board (r) og / eller Institutional Animal Care og brug Udvalg (r). Skal bortskaffes i henhold til sikkerhedsreglerne den pågældende institution besluttet på All bio-farligt affald. Kende og følge alle de rammende sikkerhed og bortskaffelse retningslinjer i hele denne procedure. <…

Representative Results

Figur 5.. Repræsentative data. A) Forventede celletal af hNPCs fastfrosset på P19, så optøet og udvidet som en Tilhænger enkeltlags hjælp enzymatisk dissociation i forhold til neurosfærer passeret via Snittemetoden. Dag 0 betegner, når cellerne blev optøet ved p20. B) Repræsentative billeder af kugler pre-chop, 10X. C)</…

Discussion

Figur 6
Figur 6.. Chopping Skematisk. Udvidelse klumpformet stamceller / stamceller i kultur ved hjælp af den mekaniske Snittemetoden.

Kritiske trin

En oversigt over snitning ekspansion paradigme er vist i figur 6.. HNPC sfære størrelse er en af de vigtige kriterier for at observere, f?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker Dr. Soshana Svendsen til kritisk gennemgang og redigering af denne rapport. Dette arbejde blev medvirket til af NIH / NINDS 1U24NS078370-01 og CIRM Dr2a-05320.

Materials

Beaker, 50 mL Fisherbrand FB-100-50 multiple manufacturers/suppliers
Bio-Safety Cabinet, class II Baker SG-603A 4 ft. or 6 ft. model.  6 ft. model recommended; multiple manufacturers/suppliers
Blades, Double-edge Prep Personna 74-0002 multiple manufacturers/suppliers.  CAUTION: Sharp
Cell Freezing Media Sigma-Aldrich C6295-50ML DMSO, serum-free
Centrifuge, swing-bucket with 15 mL inserts Eppendorf 5810 R multiple manufacturers/suppliers
Conical Tubes, 15 mL Fisherbrand S50712 multiple manufacturers/suppliers
Conical Tubes, 50 mL BD Falcon 352074 multiple manufacturers/suppliers
Controlled Rate Freezer Planer  Kryo 750 multiple manufacturers/suppliers
Cryovials, 2 mL Corning 430488 multiple manufacturers/suppliers
Culture Flask, Vented, T12.5 BD Falcon 353107 multiple manufacturers/suppliers
Culture Flask, Vented, T25 BD Falcon 353081 multiple manufacturers/suppliers
Culture Flask, Vented, T175 BD Falcon 353045 multiple manufacturers/suppliers
Culture Flask, Vented, T75  BD Falcon 353110 multiple manufacturers/suppliers
Filter, 0.22 µm, attached cup, 1 L Millipore SCGPU11RE multiple manufacturers/suppliers
Filter, 0.22 µm, attached cup, 150 mL Millipore SCGVU01RE multiple manufacturers/suppliers
Filter, 0.22 µm, attached cup, 500 mL Millipore SCGPU05RE multiple manufacturers/suppliers
Filter, 0.22 µm, attached cup, 50 mL Millipore SCGP00525 multiple manufacturers/suppliers
Filter Paper, 8.5 cm circles Whatman/GE 1001-085
Forceps, Standard Pattern – Serrated/Curved/18 cm Fine Science Tools 11001-18
Freezing Chamber, Isopropyl Alcohol Nalgene 5100-0001 "Mr. Frosty"
Incubator, 37°C/5% CO2 Forma 370 series multiple manufacturers/suppliers
Hemacytometer, Phase Hausser Scientific 1475 multiple manufacturers/suppliers
McIlwain Tissue Chopper Lafayette Instruments TC752-PD  Petri dish modification required.   CAUTION:  Moving, sharp blade.
Micropipettor, 1 – 10 μL Gilson F144562 multiple manufacturers/suppliers
Micropipettor, 100 – 1000 μL (starter kit) Gilson F167700 multiple manufacturers/suppliers
Micropipettor, 2 – 20 μL (starter kit) Gilson F167700 multiple manufacturers/suppliers
Micropipettor, 20 – 200 μL (starter kit) Gilson F167700 multiple manufacturers/suppliers
Nutdriver, Autoclavable, 5/16" Steritool 10302
Pasteur Pipets, cotton-plugged Fisherbrand 13-678-8B multiple manufacturers/suppliers
Petri Dish, Glass, Autoclavable Corning 3160-100
Pipet Aid Drummond 4-000-101 multiple manufacturers/suppliers
Shim disc McMaster-Carr VARIABLE multiple manufacturers/suppliers
Sterile barrier pipet tips, 10 μL AvantGuard AV10R-H multiple manufacturers/suppliers
Sterile barrier pipet tips, 1000 μL AvantGuard AV1000 multiple manufacturers/suppliers
Sterile barrier pipet tips, 20 μL AvantGuard AV20-H multiple manufacturers/suppliers
Sterile barrier pipet tips, 200 μL AvantGuard AV200-H multiple manufacturers/suppliers
Sterile Disposable pipettes, all-plastic wrap, 10 mL Fisherbrand 13-676-10J multiple manufacturers/suppliers
Sterile Disposable pipettes, all-plastic wrap, 2 mL Fisherbrand 13-675-3C multiple manufacturers/suppliers
Sterile Disposable pipettes, all-plastic wrap, 25 mL Fisherbrand 13-676-10K multiple manufacturers/suppliers
Sterile Disposable pipettes, all-plastic wrap, 5 mL Fisherbrand 13-676-10H multiple manufacturers/suppliers
Sterilization Pouches, 19 x 33 cm Crosstex SCL multiple manufacturers/suppliers
Strainer, 40 µm BD Falcon 352340
Tissue Culture Dishes, 60 mm BD Falcon 351007
Tube Racks, Interlocking Four-Way Fisherbrand 03-448-17
Water Bath Fisherbrand S52602Q multiple manufacturers/suppliers
Neural Progenitor Cell-Specific Processing Reagents
Neural Stem Cell Expansion Medium (Stemline) Sigma-Aldrich S3194-500ML Important to use the Stemline brand
Recombinant Human Epidermal Growth Factor (EGF) Millipore GF316 multiple manufacturers/suppliers
Recombinant Human Leukemia Inhibitory Factor (LIF) Millipore LIF1010 multiple manufacturers/suppliers
Trypan Blue (0.4%) Sigma-Aldrich T8154-100ML multiple manufacturers/suppliers
TrypLE Select (1X) Life Technologies 12563-011

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Cite This Article
Shelley, B. C., Gowing, G., Svendsen, C. N. A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue. J. Vis. Exp. (88), e51219, doi:10.3791/51219 (2014).

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