Immortalized Porcine Aortic Endothelial Cells (AOC)

CAT.NOUNITPRICE
T04481x106 cells / 1.0 ml
$0.00

Specifications


DescriptionRecently proposed as a potential model of atherosclerosis, allograft rejection and accommodation, xenotransplantation and viral haemorrhage diseases, the porcine endothelial cells provide a useful tool in studies including the molecular mechanisms of xenogeneic rejection in the swine-to-human combination, and the pathogenesis of African Swine Fever (ASF) and Classical Swine Fever (CSF). The Immortalized Porcine Aortic Endothelial Cells (AOC) is derived from stable transformation of SV40 genome into primary porcine aortic endothelial cells. The resulting AOC retains comparable surface markers to its primary cell counterparts, as well as preserving its ability to uptake acetylated low density lipoproteins (Ac-LDL) and its susceptibility to xenogeneic cell-mediated cytotoxicity (i.e. Human Natural Killer Cell-mediated lysis).
SKUT0448
SpeciesPig (Porcine)
Tissue/Organ/Organ SystemBlood Vessel
Cell MorphologyCobble-stone
Applications

For Research Use Only

Seeding Density25,000-30,000 cells/cm2; Recommended split ratio is 1:2 to 1:3
Population Doubling Time50-60 hours
Immortalization MethodTranfection with pRNS-1 plasmid encoding the SV40 genome
Cell TypeImmortalized Cells
Growth PropertiesAdherent
Expression Profile

CD29, CD31, CD41/61, CD80/86, CD46, SWC3, LAMP-1

Propagation Requirements
Use of PriCoatTMT25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells inECM-coated culture vessels unless otherwise specified in the Propagation Requirements below.

The base medium for this cell line is Prigrow II medium available at abm (TM002). To make the completed growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentration of 20% and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%. Carbon dioxide (CO2): 5%, Temperature: 37.0

Subculture Protocol

1. Subculture when the culture reaches 80-85% confluency or above.
2. Warm complete medium, Trypsin-EDTA solution, and DPBS (Ca++ and Mg++ free) to room temperature.
3. Rinse the cells with DPBS.
4. Add 2-3 ml of Trypsin-EDTA solution into flask. Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the flask in a 37°C incubator for 1 to 2 minutes or until cells completely round up. Use a microscope to monitor the change in cell morphology.
5. If needed, you may use cell scraper to scrape the cells off from the flask. Caution: make sure to only scrap the cells in one direction motion, avoid scrapping the cells back and forth with the cells scraper blade.
6. Add 6-8 ml of complete medium to the flask and transfer detached cells to the 15 ml centrifuge tube. Rinse the flask with another 3 ml of complete medium to collect the residual cells.
7. Examine the flask under a microscope for a successful cell harvest by looking at the number of cells being left behind; there should be less than 5%.
8. Centrifuge the 15 ml centrifuge tube at 200x g for 2-3 minutes. Resuspend cells in fresh complete culture medium.
9. Count and plate cells in a new prepared culture vessel.

Preservation Protocol1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO.

2. Storage Temperature: Liquid nitrogen vapour phase.
Unit quantity1x106 cells / 1.0 ml
QC

1) Flow cytometry was used to confirm the expression of endothelial cell markers such as CD29, CD31, CD41/61, CD80/86, CD46, SWC3, LAMP-1 antigen and MHC Class I antigens; 2) 51Cr release assay was used to analyse the susceptibility of AOC to xenogeneic cell-mediated cytotoxicity; 3) 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (DiI)-labelled Ac-LDL was used to evaluate Ac-LDL update by the AOC.

Disclaimer

1. For for-profit organizations and corporations, please contact [email protected] for pricing of this item.
2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at [email protected]
3. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5

FAQs


I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock).
How often do I need to change the media?
The media should be changed every 2-3 days.
Why do these cells need bio safety level II?
In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required.
Do you sell ECM coated T75 flasks?
Yes we can provide a coating service. Please inquire with [email protected]
What can I coat a larger dish to subculture?
We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html
How long can I store frozen vials for?
Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery.
Should the cap of the flask be changed before starting the cell culturing step?
No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.).
What are the markers used for characterization of these cells?
The markers used for characterization of these cells are VE cadherin, vWF and CD31.
What is the recommended storage temperature?
In general, if you received: Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen; -180C.
How is cell density crucial for drug selection?
If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don't need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic.
My cells are not detaching, what method do you recommend to trypsinize the cells?
1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope. 2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells. 3. You can try reducing the incubation time as well for coating the plate to make a thinner layer.
Why is it important to determine the optimal seeding density?
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate. If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed. If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating.
References


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  • Carrillo, A et al. "Isolation and characterization of immortalized porcine aortic endothelial cell lines" Vet Immunol Immunopathol 89(1-2):91-8 (2002). PubMed: 12208054.
  • Frandoloso, R et al. "Differences in Haemophilus parasuis adherence to and invasion of AOC-45 porcine aorta endothelial cells" BMC Vet Res. 12(9):207 (2013). PubMed: 24119995.
  • Vanier, G et al. "Characterization of the invasion of porcine endothelial cells by Streptococcus suis serotype 2" Can J Vet Res. 71(2):81-9 (2007). PubMed: 17479770.
  • Basini, G et al. "Sanguinarine inhibits VEGF-induced angiogenesis in a fibrin gel matrix" Biofactors. 29(1):11-8 (2007). PubMed: 17611290.
  • Bianco, F et al. "Angiogenic activity of swine granulosa cells: effects of hypoxia and vascularendothelial growth factor Trap R1R2, a VEGF blocker" Domest Anim Endocrinol 28(3):308-19 (2005). PubMed: 15760671.
  • Campos , E et al. "In vitro effect of classical swine fever virus on a porcine aortic endothelial cell line" Vet Res 35(6):625-33 (2014). PubMed: 15535953.
  • Crew, MD et al. "Sequence and mRNA expression of nonclassical SLA class I genes SLA-7 and SLA-8" Immunogenetics 56(2):111-4 (2004). PubMed: 15118849.
  • Johns, HL et al. "Classical swine fever virus infection protects aortic endothelial cells from pIpC-mediated apoptosis" J Gen Virol 91(pt4):1038-46 (2010). PubMed: 20007358.
  • García-Borges, CN et al. "Molecular cloning and characterization of a porcine UL16 binding protein (ULBP)-likecDNA" Mol Immunol 42(6):665-71 (2005). PubMed: 15781110.
  • Lilienfeld , BG et al. "Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity" Xenotransplantation (Københaven) 14(2):126-134 (2007). DOI: 10.1111/j.1399-3089.2007.00378.x.
  • Basini, G et al. "Catecholestogens inhibit endothelial cell growth in an angiogenesis bioassay" Endocrine Abstracts 16:618 (2008).