Name: processing of primary brain tumor tissue for stem cell assays and flow sorting
Uploaded: 2012-09-25T11:55:00
Description: Watch this Scientific Journal Video about Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting at JoVE.com

Dette arbejde blev finansieret af Ontario Institute of Cancer Research (OICR), Terry Fox Foundation og American Association of Neurologiske Surgeons.

1. Culture of Brain Tumor Tissue <ol><li> Tilsæt 200 pi optøet Liberase (Roche Applied Science) til 15 ml kunstig CSF (aCSF-se tabel 1) og anbringes i 37 ° C vandbad. Liberase TM er en blanding af proteolytiske enzymer, der anvendes til at dissociere primære vævsprøver, såvel som dyrkede tumorspheres. I modsætning trypsin-EDTA, bevarer Liberase metoden overfladeantigenet CD133. For en vævsprøve på omkring 0,5 cm 3, bruger vi 200 pi Liberase. Hvis vævet er mindre, bruger vi 100 u...

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The cancer stamceller hypotese 10, baseret på arbejde i leukæmi 21, brystkræft 11 og hjernecancer 4,5, foreslår, at kun en forholdsvis lille brøkdel af celler i tumoren, betegnet cancerstamceller, besidder en evne til omfattende proliferere og selvstændige -forny. De fleste af tumorcellerne mister evnen til at proliferere og selv-fornyelse idet de differentierer til celler, der bliver den fænotypiske signatur af tumoren. At finde de centrale celler i hjernen tumor befol...

Hjernetumorer er blandt de mest aggressive og heterogene cancere kendt hos mennesker. Selv om deres tidligere detektion og diagnose er blevet lettet af moderne neuro-imaging-teknologi, vi stadig mangler helbredende behandlinger for mange hjernetumorer, især for diffuse, invasive dem, der ligger dybt i hjernen. Hjernetumorer repræsenterer førende årsag til kræft dødelighed hos børn på grund af deres meget aggressive og ofte uhelbredelige karakter. Glioblastoma (GBM), den mest almindelige primær hjernetumor i voksne, er en af de mest aggressive humane cancere, frygtede for sin ensartet fatal prognose 1. Denne yderst ondartet astrocytisk tumor (WHO grad 4) forekommer normalt i de cerebrale hemisfærer af voksne, og kan også forekomme hos små børn og spædbørn. Dens vækst er hurtig og infiltrativ, og diagnostiske patologiske funktioner omfatter nuklear pleomorphism, mikrovaskulær proliferation og nekrose 2,3. For adults med nydiagnosticeret GBM, median overlevelse sjældent strækker sig ud over 12 måneder 1, med generelt dårlige svar på alle terapeutiske modaliteter. Vi bemærkede, at der er mange funktionelle og genetiske ligheder fælles for somatiske stamceller og cancerceller, og at de molekylære veje, der regulerer normal udvikling af hjernen ofte dysreguleret i cancer. Ved anvendelsen stamcellebiologi paradigmer til studiet af hjernesvulster, var vi de første forskere til prospektivt identificere og oprense en subpopulation af celler fra menneskelige GBMS som udviste på stamcelle egenskaber spredning, selvfornyelse og differentiering in vitro 4 og i vivo 5. Vi anvendte dyrkningsbetingelser og assays oprindeligt anvendes til at karakterisere normale, neurale stamceller (NSC) in vitro 6,7 til flere pædiatriske og voksne hjernetumorer, og beriget med disse stilk-lignende celler ved cellesortering til den neurale progenitorcelle overflademarkør CD133 8 ,9. The CD133 + hjernetumor fraktion indeholdt celler, der havde en meget højere frekvens af tumorinitiering end CD133-fraktionen i NOD-SCID musehjerner 5,10. Dette formelt fastslået, at kun en sjælden delmængde af hjernetumorceller med stamceller egenskaber er tumorfremkaldende, tjener dem navnet &quot;brain tumorfremkaldende celler&quot; eller &quot;BTICs&quot;. Den hidtil ukendte identifikation af BTICs giver nye indsigter i den menneskelige hjerne tumorigenese, der giver en kraftig støtte til kræft stamceller hypotese 10-13 som grundlag for mange solide tumorer, og etablerer en ny cellulære mål for mere effektive behandlinger mod kræft 14-20. Terapier, der fokuserer på at dræbe hovedparten af ​​tumoren kan gå glip af sjældne stilk-lignende fraktion, således at tumoren fortsat at vokse. Terapier, der fokuserer på at dræbe kræft stamceller kan give bedre behandling og prognose for patienter med hjernetumorer. For at undersøge BTIC populationer har vi forfinet vores culture protokoller til specifikt selekterer for cellepopulationer inden humane hjernetumorer, der besidder stamceller egenskaber. Serumfrit, neural stamcelle (NSC) medium giver mulighed for opretholdelse af en udifferentieret stamcelle tilstand, og tilsætningen af ​​basisk fibroblastvækstfaktor (bFGF), epidermal vækstfaktor (EGF) og leukæmiinhiberende faktor (LIF) tillader udbredelsen af ​​multi-potent, selvfornyende, og udvidelige menneskelige tumorspheres. Her beskriver vi de metoder, der er involveret i behandling af primære hjernetumorer og dyrkning dem i NSC medium til at berige BTIC befolkninger. Vi har kaldt vores eksperimentelle modelsystem &quot;BTIC patientisolater&quot; for at understrege, at disse celler er kun minimalt dyrkes under stamceller celle betingelser med henblik på selektion stamcellepopulationer. Efterfølgende immunolabelling for BTIC populationer for vigtige stamceller markører såsom CD133 og CD15 og flowcytometri-analyse er også beskrevet. Vi så diskutere begrænsende fortynding analyse,som hjælper med at studere selvfornyelse potentiale BTICs. Endelig udnytter vi den genekspressionsanalyse af disse sjældne celler ved at sortere enkelte celler på AmpliGrid objektglas og udføre enkelt celle RT-PCR. Disse teknikker gælder også for andre hjernetumorer, såsom medulloblastom ependymoma og pædiatriske gliomer.

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> 01:01 DMEM: F12 </td><td> Invitrogen </td><td> 11320-082 </td></tr><tr><td> N2 supplement </td><td> Invitrogen </td><td> 17502-048 </td></tr><tr><td> 1M HEPES </td><td> Wisent </td><td> 330-050-EL </td></tr><tr><td> Glucose </td><td> Invitrogen </td><td> 15023-021 </td></tr><tr><td> N-acetylcystein </td><td> Sigma Aldrich </td><td> A9165-25g </td></tr><tr><td> Neural overlevelse faktor -1 (NSF-1) </td><td> Lonza Clonetics </td><td> CC-4323 </td></tr><tr><td> Epidermal vækstfaktor (EGF) </td><td> Sigma Aldrich </td><td> E9644 </td></tr><tr><td> Basisk fibroblastvækstfaktor (bFGF) </td><td> Invitrogen </td><td> PHG0261 </td></tr><tr><td> Leukæmiinhiberende faktor (LIF) </td><td> Millipore </td><td> LIF1010 </td></tr><tr><td> Antibiotikum / mykotisk </td><td> Wisent </td><td> 450-115-EL </td></tr><tr><td> Liberase TM </td><td> Roche </td><td> 05 401 119 001 </td></tr><tr><td> Ammoniumchloridopløsning </td><td> Stem Cell Technologies </td><td> 07.850 </td></tr></tbody></table>

processing of primary brain tumor tissue for stem cell assays and flow sorting

Hjernetumorer omfatter typisk morfologisk forskellige celler, som udtrykker en række neurale afstamning markører. Kun en forholdsvis lille del af cellerne i tumoren med stamceller egenskaber, betegnet hjernesvulst initierende celler (BTICs), besidder en evne til at differentiere langs flere slægter, selv-fornyelse og initiere tumorer in vivo. Vi anvendte dyrkningsbetingelser oprindeligt anvendt til normale, neurale stamceller (NSC) og en række humane hjernetumorer og fandt, at denne dyrkningsmetode specifikt selekterer for stilk-lignende populationer. Serumfrit medium (NSC) giver mulighed for opretholdelse af en udifferentieret stamcelle tilstand og tilsætning af bFGF og EGF muliggør proliferationen af ​​multi-potente, selvfornyende, og ekspanderbare tumorspheres. For yderligere at karakterisere hver tumor BTIC population, vi evaluere celleoverflademarkører ved flowcytometri. Vi kan også sortere populationer af interesse for mere specifikke characterization. Selvfornyelse analyser udføres på enkelte BTICs sorteret i 96-brønds plader, dannelsen af ​​tumorspheres efter inkubation ved 37 ° C indikerer tilstedeværelsen af ​​en stam-eller progenitorcelle. Multiple celleantal i en bestemt population kan også sorteres i forskellige brønde for begrænsende fortynding analyse for at analysere selvfornyelse kapacitet. Vi kan se differentiel genekspression i en bestemt cellepopulation ved hjælp af enkeltcelle-RT-PCR. De følgende protokoller beskriver vores procedurer for dissociation og dyrkning af primære humane prøver at berige for BTIC populationer samt dissociationen af ​​tumorspheres. Også indbefattet er protokoller for farvning for flowcytometrianalyse eller sortering, selvfornyelse assays, og enkeltcelle-RT-PCR.

Watch this Scientific Journal Video about Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting at JoVE.com

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Identifikationen af ​​hjernesvulst initierende celler (BTICs), de sjældne celler i en heterogene tumor besidder stamceller egenskaber, giver ny indsigt i den menneskelige hjerne tumor patogenese. Vi har forfinet særlige dyrkningsbetingelser at berige for BTICs, og vi rutinemæssigt bruger flowcytometri til yderligere berige disse befolkningsgrupper. Selvfornyelse assays og transkript analyse ved enkelt celle RT-PCR kan efterfølgende udføres på disse isolerede celler.

Behandling af primær hjernetumor Tissue for stamcelle Analyser og Flow Sortering

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Research

JoVE Journal

Medicine

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Fremstilling af myeloide afledte suppressorceller (MDSC) fra naive og pancreas Tumorbærende mus under anvendelse af flowcytometri, og Automatiseret magnetisk aktiveret cellesortering (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolering af cardiomyocyte Kerner fra Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Isolation, karakterisering og sammenlignende differentiering af humane Dental Pulp stamceller fra blivende tænder ved hjælp af to forskellige metoder

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Afledt Cellekultur og isolering af CD133<sup&gt; +</sup&gt; Formodede kræftstamceller fra melanom

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

A Novel Høj opløsning<em&gt; In vivo</em&gt; Imaging teknik til at undersøge den dynamiske respons af Intrakranielle Structures til tumorvækst og Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Tilpasning af halvautomatiseret Cirkulerende Tumor Cell (CTC) analyser til kliniske og prækliniske forskning applikationer

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Evaluering af Stem Cell Properties i Human ovariekarcinomceller Brug Multi og enkelt celle-baserede Spheres Analyser

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

Primær tumor og MEF Cell Isolation at studere Lung Metastase

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

I Vitro Assays at vurdere blod-hjerne barriere mesh-lignende fartøj dannelse og afbrydelse

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

Prøvetagningsmetoder og forarbejdning af Biobank vævsprøver fra svin biomedicinsk modeller

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...