| 產(chǎn)品名稱 | 
      TIME | 
    
    
      | 商品貨號(hào) | 
      B244169 | 
    
      
      | Organism | 
      Homo sapiens, human | 
    
      
      | Tissue | 
      Foreskin; Dermal microvascular endothelium | 
    
      
      | Cell Type | 
      Endothelial cells immortalized with hTERT | 
    
      
      | Product Format | 
      frozen | 
    
      
      | Morphology | 
      Endothelial-like | 
    
      
      | Culture Properties | 
      Adherent | 
    
      
      | Biosafety Level | 
      2 [Cells contain EMCV viral DNA sequences]
Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.   | 
    
      
      | Disease | 
      Normal | 
    
      
      | Age | 
      Neonatal | 
    
      
      | Gender | 
      Male | 
    
      
      | Applications | 
      The cells represent an effective cell model for studying endothelial cell biology including signal transduction and angiogenesis.  | 
    
      
      | Storage Conditions | 
      Liquid nitrogen vapor phase | 
    
      
      | Karyotype | 
      This is a diploid cell line of male origin with a modal chromosome number of 46 and a low rate of polyploidy. The line shows some karyotypic instability at later passages. | 
    
      
      | Images | 
        | 
    
      
      | Derivation | 
      The telomerase-immortalized human microvascular endothelium cell line, TIME, was derived from a primary culture of neonatal foreskin microvascular endothelial cells (HMVEC) of the dermis.  
The primary HMVECs were immortalized by infection with the retrovirus WZLblast3:hTERT and cultured in complete growth medium containing blasticidin.   | 
    
      
      | Antigen Expression | 
      Positive for integrin alpha v beta 3 Ref  Venetsanakos E, et al. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells. Exp. Cell Res. 273(1):21-33,  2002. PubMed: 11795943 and CD31 (flow cytometry) Ref  Venetsanakos E, et al. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells. Exp. Cell Res. 273(1):21-33,  2002. PubMed: 11795943
 
  | 
    
      
      | Receptor Expression | 
      The cells express the low density lipoprotein (LDL) receptor and are capable of acetylated LDL uptake. Ref  Venetsanakos E, et al. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells. Exp. Cell Res. 273(1):21-33,  2002. PubMed: 11795943 
 | 
    
      
      | Comments | 
      The immortalized cells do not undergo growth arrest in culture due to the exogenous hTERT expression. 
When plated on Matrigel, TIME cells undergo tubule formation exhibiting capillary-like structures.  | 
    
      
      | Complete Growth Medium | 
      The base medium for this cell line is Vascular Cell Basal Medium (ATCC® PCS-100-030), supplemented with Microvascular Endothelial Cell Growth Kit-VEGF (ATCC® PCS-110-041) and 12.5 μg/mL blasticidine.  | 
    
      
      | Subculturing | 
      Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 
Note: Subculture when cultures are about 80% confluent. 
    - Prior to subculturing, determine the number of flasks needed. Add the appropriate volume of medium to each flask and allow the flasks to equilibrate in a 37°C, 5% CO2, humidified incubator for at least 30 minutes. If not using vented caps, loosen caps of flasks.
 
    - Remove and discard spent medium.
 
    - Briefly rinse the cells with Dulbecco's Phosphate Buffered Saline (D-PBS, ATCC 30-2200) and discard rinse solution.
 
    - Add 2.0 to 3.0 mL room temperature Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) to the flask. Incubate at 37°C for 5 min (until cells have detached).
 
    - Neutralize trypsin by adding an equal volume of room temperature 2% FBS in D-PBS.
 
    - Transfer cells to a centrifuge tube. Rinse the flask with an additional room temperature 2% FBS in D-PBS and pool into centrifuge tube with cells.
 
    - Centrifuge cells at 250 x g for 10 min at room temperature.
 
    - Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
 
    - Count cells, and seed 5 x 103 to 8 x 103 viable cells/cm2 to new culture vessels. Subculture when cells become 80 to 90% confluent, which normally yield approximately 3.0 x 104 viable cells/cm2.
 
    - Incubate cultures at 37°C in a 5% CO2 humidified incubator.
 
 
Subcultivation ratio: A subcultivation ratio of 1:4 to 1:6 is recommended. 
Medium renewal: Every 2 to 3 days 
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney.  | 
    
      
      | Cryopreservation | 
      Fetal bovine serum, 90% (v/v); DMSO, 10% (v/v). Cell culture tested DMSO is available as ATCC Catalog No. 4-X. 
 
  | 
    
      
      | Culture Conditions | 
      Temperature: 37°C Atmosphere: Air, 95%; Carbon dioxide (CO2), 5%  | 
    
      
      | STR Profile | 
      D5S818: 11 D13S317: 9, 11  D7S820: 8, 9  D16S539: 9, 12 vWA:  16, 18 THO1: 6, 7 TPOX: 8 CSF1PO: 11, 12 Amelogenin:  XY   | 
    
      
      | Population Doubling Level (PDL) | 
      As part of our quality control, we have tested this cell line for its ability to grow for a minimum of 15 population doublings after recovery from cryopreservation. We have also compared its karyotype, telomerase expression level, growth rate, morphology and tissue-specific markers when first recovered from cryopreservation with that of cells at 10+ population doublings to ensure that there is no change in these parameters and that the cells are capable of extended proliferation. | 
    
      
      | Name of Depositor | 
      M McMahon | 
    
      
      | Year of Origin | 
      June 2001 | 
    
      
      | References | 
      
            Venetsanakos E, et al. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells. Exp. Cell Res. 273(1):21-33,  2002. PubMed: 11795943
         
    
        
            Lagunoff M, et al. De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells. J. Virol. 76(5):2440-2448, 2002. PubMed:  11836422
         
    
        
            Yi X, et al. Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels.  Mol. Cell. Biol. 19(6): 3989-3997, 1999. PubMed: 10330139
         
    
        
            Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells.  Science 279: 349-352, 1998. PubMed: 9454332
         
    
        
            Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988
         
    
        
            Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 4th edition. New York: Wiley Liss; 2000. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 10. 
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