Human iPSC-Derived Endothelial Colony Forming Cells
| Catalog. No. | Product Name | Product Quantity | Storage on Arrival | Thawing Instructions | Storage Once Thawed |
| ax2015 | Human iPSC-Derived Endothelial Colony Forming Cells (Male) | 1 million cells/vial | Liquid Nitrogen | Follow protocol | N/A |
| ax2015 | Human iPSC-Derived Endothelial Colony Forming Cells (Female) | 1 million cells/vial | Liquid Nitrogen | Follow protocol | N/A |
| ax2030 | Endothelial Colony Forming Cell Culture Medium | 500 mL | Aliquot and store at -80°C for up to 6 months | Thaw at 4°C or at room temperature | Store at 4°C for up to 1 week |
| ax0049 | Fibronectin Coating Solution | 1 mL | Aliquot and store at -80°C for up to 3 months | Thaw at 4°C | Once diltued, use immediately |
| ax0044 | Unlock | 25 mL | Aliquot & store at -80°C for up to 6 months | Thaw at 4°C | Store at 4°C for up to 1 week |
| ax2215 | Human iPSC-Derived Endothelial Colony Forming Cell Kit (Male) |
Kit Components
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See above for component details | See above for component details | See above for component details |
| ax2219 | Human iPSC-Derived Endothelial Colony Forming Cell Kit (Female) |
Kit Components
|
See above for component details | See above for component details | See above for component details |
Recommendations
- Recommended culture vessel coating: Fibronectin
- Recommended cell culture medium: Endothelial Colony Forming Cell Culture Medium
- Recommended seeding density for assay: 10,000 viable cells/cm2
- Recommended centrifugation speed: 300 x g for 5 minutes
Important! Always count the number of viable cells after thawing.
Important! Axol Endothelial Colony Forming Cell Culture Medium DOES NOT contain antibiotics or antifungal agents. Axol Bioscience does not recommend the use of antimicrobial agents such as penicillin, streptomycin and amphotericin.
Antimicrobial agents should not be necessary if proper aseptic technique is adopted
Coating
- Dilute the stock Fibronectin Coating Solution 1:100 in sterile water to make 1x working solution e.g. 100 μL in 10 mL.
- On the day prior to thawing the cells, coat the surface of your culture vessel with the Fibronectin 1x working solution.
- We recommend coating at a volume of 200 μL per cm2 however, please optimize for your experiments.
- Incubate the culture vessel overnight at 37°C in a humidified incubator.
Thawing and Plating
- Having coated the culture vessel the day before, proceed with thawing the cells.
- Transfer the vial of cells from liquid nitrogen storage with the vial buried in dry ice. Remove the vial from dry ice and transfer it immediately to a 37°C water bath.
- Thaw the cells quickly in a 37°C water bath. Remove the vial before the last bit of ice has melted, after ~1-2 minutes.
- Wipe the outside of the vial with 70% ethanol.
- Gently resuspend the cells and transfer to a 15 mL sterile conical tube.
- Slowly add 10 mL of pre-warmed, 37°C, Endothelial Colony Forming Cell Culture Medium.
- Rinse the cryovial with 1 mL of Endothelial Colony Forming Cell Culture Medium to ensure all of the cells are
transferred. - Centrifuge the cells at 300 x g for 5 minutes. Carefully remove the supernatant and resuspend the cell pellet in
1-2 mL of pre-warmed, 37°C, Endothelial Colony Forming Cell Culture Medium. - Perform a cell count to determine the number of viable cells.
- Dilute the cells into the required volume of pre-warmed, 37°C, Endothelial Colony Forming Cell Culture Medium.
- Seed cells into the pre-coated culture vessel at the recommended seeding density of 10,000 viable cells/cm2.
- Incubate the cells at 37°C, 5% CO2 in a humidified incubator.
- Frequency of media changes: Every 2 days
Passaging
- Passage when the culture reaches: 80-90% confluent
- Recommended passaging reagent: Unlock l On the day prior to passaging the cells, coat the culture vessel with Fibronectin 1x working solution and incubate overnight at 37°C in a humidified incubator.
- Remove all spent culture medium from the cell culture vessels.
- Gently rinse the surface of the cell layer once with PBS, 2 mL of PBS per 10 cm2 culture surface area. Discard the PBS.
- Add 1 mL per 10 cm2 of culture surface area of cold/room temperature Unlock passaging reagent. Evenly distribute it over the entire cell layer.
- Incubate the cells for 5 minutes at 37°C. Observe the cells at regular intervals for detachment from the culture vessel.
- Once the cells have detached, dilute out the passaging reagent with four volumes pre-warmed, 37°C, Endothelial Colony Forming Cell Culture Medium. For example, if 1 mL of Unlock is used, then add 4 mL of the medium to stop the reaction.
- Transfer the cell suspension to a sterile conical tube.
- Centrifuge the cells at 300 x g for 5 minutes.
- Carefully remove the supernatant and resuspend the cell pellet in 1-2 mL of pre-warmed, 37°C, Endothelial Colony Forming Cell Culture Medium.
- Perform a cell count to determine the number of viable cells.
- Dilute the cells into the required volume of pre-warmed, 37°C, Endothelial Colony Forming Cell Culture Medium.
- Seed cells into the pre-coated culture vessel at the recommended seeding density of 10,000 viable cells/cm2.
- Incubate the cells at 37°C, 5% CO2 in a humidified incubator.
Usage Statement
Our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans.
