rat_fetal_nsc_man.pdf

User ManualCorporate HeadquartersInvitrogen Corporation1600 Faraday AvenueCarlsbad,CA 92008T:1 760 603 7200F:1 760 602 6500E:tech_For country-specific contact information visit our web site at Rat Fetal Neural Stem Cells Catalog nos.N7744-100,N7744-200 Rev.date:7 April 2009 Manual part no.A11229 MAN0001642 ii iiiTable of Contents Contents and Storage.iv Additional Products.v Introduction.1 Rat Fetal Neural Stem Cells(NSCs).1 Phenotype Marker Expression of Rat Fetal NSCs.3 Methods.5 Handling Rat Fetal NSCs.5 Media Requirements.6 Preparing Complete StemPro NSC SFM.7 Preparing Matrix for Adherent Cell Culture.8 Thawing and Establishing Cells.10 Subculturing Cells.12 Subculturing Cells(Adherent Culture).13 Subculturing Cells(Suspension Culture).14 Freezing Cells.15 Differentiating Rat Fetal NSCs.17 Characterizing Phenotype of Rat Fetal NSCs.18 Troubleshooting.20 Appendix.22 Recipes.22 Technical Support.23 Purchaser Notification.24 References.26 Contents and Storage Kit Configurations Catalog no.R7744-100 includes cells only.Catalog no.R7744-200 includes cells plus media.Shipping Rat Fetal Neural Stem Cells,StemPro NSC SFM Supplement,FGF Basic Recombinant Human,and EGF Recombinant Human are shipped on dry ice.KnockOut DMEM/F-12 is shipped at room temperature.Kit Contents and Storage Kit components and storage conditions for R7744-100 and R7744-200 are listed in the table below.R7744-100 Amount Storage Rat Fetal Neural Stem Cells(2 106 cells/mL in freezing medium*)1 mL Liquid nitrogen R7744-200 Amount Storage Rat Fetal Neural Stem Cells(2 106 cells/mL in freezing medium)1 mL Liquid nitrogen KnockOut DMEM/F-12 500 mL 2 to 8C,in the dark StemPro NSC SFM Supplement 10 mL 5 to 20C,in the dark FGF Basic Recombinant Human 10 g 2 to 8C EGF Recombinant Human 10 g 2 to 8C*Freezing medium:DMEM/F-12 containing non-essential amino acids,2 mM GlutaMax,0.1 mM-mercaptoethanol,100 g/ml apo-transferrin,25 g/ml insulin,100 M putrescine,30 nM sodium selenite,20 nM progesterone,10 ng/ml bFGF,plus 10%DMSO.Handle cells as potentially biohazardous material under at least Biosafety Level 1(BL-1)containment.This product contains Dimethyl Sulfoxide(DMSO),a hazardous material.Review the Material Safety Data Sheet(MSDS)before handling.Material Safety Data Sheets(MSDSs)are available on our website at vAdditional Products Additional Products The products listed in this section may be used with Rat Fetal Neural Stem Cells.For more information,refer to our website()or contact Technical Support(see page 22).Item Quantity Cat.no.StemPro NSC SFM(contains KnockOut DMEM/F-12,FGF Basic Recombinant Human,EGF Recombinant Human,and StemPro NSC SFM Supplement)1 kit A1050901 GlutaMAX-I Supplement 100 mL 35050-061 KnockOut DMEM/F-12 500 mL 12660-012 FGF Basic Recombinant Human(bFGF)10 g PHG0024 EGF Recombinant Human 10 g PHG0314 Fetal Bovine Serum(FBS),ES Cell-Qualified 100 mL 500 mL 16141-061 16141-079 BSA,10%Stock Solution 25 mL P2489 Dulbecco s Phosphate Buffered Saline(D-PBS),containing no calcium,magnesium,or phenol red 500 mL 14190-144 Dulbecco s Phosphate Buffered Saline(D-PBS),containing calcium and magnesium,but no phenol red 500 mL 14040-133 Dulbecco s Modified Eagle Medium(D-MEM)(1X),liquid(high glucose)1000 mL 11995-040 CELLStart Defined,Humanized Substrate for Cell Culture 2 mL 10142-01 Geltrex Reduced Growth Factor Basement Membrane Matrix 5 mL 12760-021 Fibronectin,Human Plasma 5 mg 33016-015 StemPro Accutase Cell Dissociation Reagent 100 mL A11105-01 Neurobasal Medium(1X),liquid 500 mL 21103-049 B-27 Serum-Free Supplement(50X),liquid 10 mL 17504-044 N-2 Supplement(100X),liquid 5 mL 17502-048-Mercaptoethanol(1,000X),liquid 50 mL 21985-023 Continued on next page vi Additional Products,continued Additional Products,continued The products listed in this section may be used with Rat Fetal Neural Stem Cells.For more information,refer to our website()or contact Technical Support(see page 22).Item Quantity Cat.no.Trypan Blue Stain 100 mL 15250-061 LIVE/DEAD Cell Vitality Assay Kit 1000 assays L34951 Countess Automated Cell Counter(includes 50 Countess cell counting chamber slides and 2 mL of Trypan Blue Stain)1 unit C10227 Water,distilled 20 100 mL 15230-196 Products for Marker Analysis The products listed below may be used for analyzing the phenotype of undifferentiated Rat Fetal Neural Stem Cells,and well as neurons,oligodendrocytes,and astrocytes.In addition to the primary antibodies listed below,Invitrogen offers a variety of isotype specific secondary antibodies conjugated with enzymatic and fluorescent indicators,as well as antibody sera and diluents.For more information,refer to our website()or contact Technical Support(see page 22).Item Quantity Cat.no.Mouse anti-MAP2 100 g 13-1500 Rabbit anti-Doublecortin(Dcx)100 g 48-1200 Mouse anti-A2B5(105)100 g 433110 Rabbit anti-GFAP(Glial Fibrillary Acid Protein)-concentrate 1 mL 18-0063 DAPI(4,6-diamidino-2-phenylindole,dihydrochloride)10 mg D1306 ProLong Gold Antifade Reagent 10 mL P36930 ProLong Gold Antifade Reagent with DAPI 10 mL P36931 1Introduction Rat Fetal Neural Stem Cells(NSCs)Introduction Rat Fetal Neural Stem Cells(NSCs)are isolated from the cortexes of the fetal(embryonic day 14)Sprague-Dawley rats.The cells are isolated under sterile conditions and expanded in N-2/DMEM/F-12 medium supplemented with 10 ng/mL basic fibroblast growth factor(bFGF)before cryopreservation at passage 0(P0)in 90%N-2/DMEM/F-12 medium and 10%DMSO.Each vial of Rat Fetal NSCs contains 2 106 cells/mL that can be expanded in culture for up to three passages.Withdrawal of bFGF allows the cells to differentiate into neurons,astrocytes,and oligodendrocytes(Gage,2000;Wu et al.,2002).Because of their property to generate glial cells and electrically active neurons,Rat Fetal NSCs can be used for neuroscience studies as well as stem cell differentiation,tissue engineering,cell and genetic therapy,and transplantation experiments(Bjorklund&Lindvall,2000;Gage,2000;Temple,2001;Zhao et al.,2008).We recommend that you use StemPro NSC SFM(see page v)for optimal growth and expansion as adherent cells on matrix or suspended neurospheres.Characteristics of Rat Fetal NSCs Isolated from fetal brain cortex of Sprague-Dawley rats on day 14 of gestation(E14)Capacity for self-renewal Ability to differentiate into neurons,oligodendrocytes,and astrocytes Stain positive for the neural stem cell-type specific marker nestin(75%)Stain 10%for differentiated phenotype markers Dcx,GFAP,and GalC Exhibit a doubling time of 2030 hours,which tends to increase with passage number Can be expanded in culture up to three passages without differentiation Continued on next page Rat Fetal NSCs,continued Rat Fetal NSC Culture Primary cells isolated from the cortical neuroepithelium of the fetal(embryonic day 14)rat can be expanded up to three passages in culture.The image below shows undifferentiated Rat Fetal NSCs maintained as an adherent culture.Figure 1.Bright field image of adherent Rat Fetal NSCs at P3(passage 3)that have been cultured in complete StemPro NSC SFM for 10 days.The image was captured using 10X objective lens.Continued on next page 2 Phenotype Marker Expression of Rat Fetal NSCs Undifferenti-ated Rat Fetal NSCs The presence of basic fibroblast growth factor(bFGF)in complete StemPro NSC SFM allows the maintenance of Rat Fetal NSCs in their undifferentiated state.The images below show the phenotype marker expression of undifferentiated Rat Fetal NSCs after three rounds of passaging(P3)in StemPro NSC SFM.A B C D Figure 2.Fluorescence images(20X)of Rat Fetal NSCs at P3 that have been cultured in StemPro NSC SFM for 10 days and then stained for the appropriate phenotypic marker.Cells were stained for the undifferentiated NSC marker,nestin(red)(panel A),for the neuronal marker,Dcx(red)(panel B),for the oligodendrocyte marker,GalC(green)(panel C),or for the astrocyte marker,GFAP(red)(panel D).The nuclei were stained with DAPI(blue)in all panels.While approximately 90%of the cells stain positive for the undifferentiated NSC marker,nestin,less than 10%of the cells are positive for differentiated cell type markers Dcx,GalC,and GFAP.Continued on next page 3Phenotype Marker Expression of Rat Fetal NSCs,continued Differentiated Rat Fetal NSCs Rat Fetal NSCs spontaneously differentiate into neurons,oligodendrocytes,or astrocytes upon withdrawal of bFGF from culture media,or they can be enriched toward a specific lineage upon selection on differentiation medium.The images below show the differentiation potential of Rat Fetal NSCs after three passages in StemPro NSC SFM.A B C D Figure 3.Fluorescence images(20X)of Rat Fetal NSCs that have been cultured in StemPro NSC SFM for three passages,and then allowed to differentiate into neurons,oligodendrocytes,or astrocytes.Upon differentiation,cells start to lose undifferentiated NSC marker,nestin,but stain positive for differentiated cell type markers Dcx,GalC,and GFAP.Cells were stained for the undifferentiated NSC marker,nestin(red)(panel A),for the neuronal marker,Dcx(red)(panel B),for the oligodendrocyte marker,GalC(green)(panel C),or for the astrocyte marker,GFAP(red)(panel D).The nuclei were stained with DAPI(blue)in all panels.4 Methods Handling Rat Fetal NSCs As with other mammalian cell lines,when working with Rat Fetal NSCs,handle as potentially biohazardous material under at least Biosafety Level 1(BL-1)containment.For more information on BL-1 guidelines,refer to Biosafety in Microbiological and Biomedical Laboratories,5th ed.,published by the Centers for Disease Control,or see the following website:www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm Guidelines for Culturing Rat Fetal NSCs Follow the general guidelines below to grow and maintain Rat Fetal NSCs.All solutions and equipment that come in contact with the cells must be sterile.Always use proper aseptic technique and work in a laminar flow hood.Before starting experiments,make sure that the cells have been established(at least 1 passage).For consistent results in your differentiation studies and other experiments,we recommend using cells below passage 3(P3).If you expand Rat Fetal NSCs beyond P3,we recommend that you perform another round of characterization prior to further experiments.For general maintenance of Rat Fetal NSCs in adherent culture,the growth rate should be in mid-logarithmic phase with 7590%confluency prior to subculturing.Passage cells at a seeding density of 50,000 cells/cm2.Note:Passaging Rat Fetal NSCs at a lower density causes the cells to differentiate.In suspension culture as neurospheres,Rat Fetal NSCs can be passaged when neurospheres are larger than 3.5 mm in diameter.When thawing or subculturing cells,transfer cells into pre-warmed medium.Standard physical conditions for Rat Fetal NSCs grown in StemPro NSC SFM are 36 to 38C in a humidified atmosphere of 4 to 6%CO2 in air.Continued on next page 5Media Requirements?It is very important to strictly follow the guidelines for culturing Rat Fetal Neural Stem Cells in this manual to keep them undifferentiated.Media Requirements We recommend using complete StemPro NSC SFM for optimal growth and expansion of Rat Fetal NSCs,and to keep them undifferentiated.StemPro NSC SFM is designed to support growth of neural stem cells derived from embryonic stem cells or isolated from fetal tissue,which can be grown either as suspended neurospheres or as adherent culture on CELLStart,fibronectin,or poly-L-ornithine coated tissue culture treated vessels(see page v for ordering information).Note:If cultured in serum containing medium,the cells might require adaptation to serum-free medium(see page 12).Prepare your growth medium prior to use.To maintain undifferentiated NSCs,supplement the medium every day with bFGF to 10 ng/mL.Note:If you are using complete StemPro NSC SFM to culture your cells,you do not need to supplement the medium with bFGF.When thawing or subculturing cells,transfer cells into pre-warmed medium at 37C.We recommend that you aliquot complete growth medium into required working amounts to avoid exposing it to 37C multiple times.You may store the complete StemPro NSC SFM in the dark at 4C for up to four weeks.Do not freeze complete StemPro NSC SFM.You may refreeze unused StemPro NSC SFM Supplement;however,avoid repeated freeze-thaw cycles.6 7Preparing Complete StemPro NSC SFM Preparing Complete StemPro NSC SFM StemPro NSC SFM complete medium consists of KnockOut D-MEM/F-12 with StemPro NSC SFM Supplement,EGF,bFGF,and GlutaMAX-I.Complete medium is stable for up to 4 weeks when stored in the dark at 4C.To make 100 mL of complete StemPro NSC SFM,aseptically mix the following components:Component Concentration Amount KnockOut D-MEM/F-12 1X 97 mL GlutaMAX-I Supplement 2 mM 1 mL bFGF 20 ng/mL 2 g EGF 20 ng/mL 2 g StemPro NSC SFM Supplement 2%2 mL Note:You may observe a white precipitate when thawing StemPro NSC SFM,which will disappear when it is completely thawed or dissolved.8 Preparing Matrix for Adherent Cell Culture Coating Culture Vessels for Adherent Cell Culture If you prefer to maintain your Rat Fetal NSCs as an adherent culture,you may use CELLStart,fibronectin,or poly-L-ornithine.The attachment strength of Rat Fetal NSCs is greatest for CELLStart,followed by fibronectin,and is weakest for poly-L-ornithine.Using CELLStart as matrix:1.Dilute CELLStart 1:100 in D-PBS with calcium and magnesium(i.e.,50 L of CELLStart into 5 mL of D-PBS)(see page v).2.Coat the surface of the culture vessel with the working solution of CELLStart(14 mL for T75,7 mL for T25,3.5 mL for 60-mm dish,2 mL for 35-mm dish).3.Incubate the culture vessel at 37C in a humidified atmosphere of 5%CO2 in air for 1 hour.4.Remove the vessel from the incubator and store until use.Immediately before use,remove all CELLStart solution and replace with complete StemPro NSC SFM.Note:You may coat the plates in advance,and store at 4C wrapped tightly with Parafilm for up to 2 weeks.Do not remove CELLStart solution until just prior to use.Make sure the plates do not dry out.Using fibronectin as matrix:1.Dilute fibronectin(see page v)in distilled water to make 1 mg/mL stock solution.Store at 20C.2.Dilute fibronectin stock solution 1:50 in PBS(see page v)to make 20 g/mL working solution.Store at 20C until use.3.Coat the surface of the culture vessel with the working solution of fibronectin(14 mL for T75,7 mL for T25,3.5 mL for 60-mm dish,2 mL for 35-mm dish).4.Incubate the culture vessel at 37C in a humidified atmosphere of 5%CO2 in air for 1 hour.5.Remove the vessel from the incubator and store until use.Immediately before use,remove all fibronectin solution and replace with complete StemPro NSC SFM.Note:You may coat the plates in advance,and store at 4C wrapped tightly with Parafilm for up to 2 weeks.Do not remove fibronectin solution until just prior to use.Make sure the plates do not dry out.Continued on next page 9Preparing Matrix for Adherent Cell Culture,continued Coating Culture Vessels for Adherent Cell Culture,cont