RFP Stably Expressing Enhanced Human Primary Hepatocytes

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RFP Stably Expressing Enhanced Human Primary Hepatocytes

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Cat.No.: T3939
Quantity: 5 x 106 cells/1.0ml
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Email: order@abmGood.com
Phone: 604-247-2416 (Local), 1-866-757-2414 (Toll free)
Fax: 604-247-2414
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Purchase Cell Line Insurance with your immortalized or stable cell line order and have peace of mind that abm will provide an additional vial should you require a replacement, no claim form needed or questions asked!

4 Month Coverage Plan
Cat. No. C192
Coverage Cells for four months from delivery of original order.
Cost $500 per cell line ordered for
Credit Options - If a replacement vial is not requested during the first four months, your insurance fees will be turned into a credit that can be applied towards any Cell Culture reagents.
- Credit valid for six months after insurance expires.

6 Month Coverage Plan
Cat. No. C193
Coverage Cells for six months from delivery of original order.
Cost $650 per cell line ordered for
Credit Options - If a replacement vial is not requested during the first six months, your insurance fees will be turned into a credit that can be applied towards any Cell Culture reagents.
- Credit valid for six months after insurance expires.
Notes:
* Please inquire prior to order placement. Not valid as Add-On after original order has been shipped.
** Insurance and/or credit does not include shipping and this is to be arranged by the customer


***Live cell shipment is available upon request for an additional $95.00 fee; please include Cat# C088 when placing your order to receive this service.***

*For for-profit organizations and corporations, please contact quotes@abmgood.com for pricing of this item.

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 technical@abmgood.com.
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*Notes about Promo Codes: For NON-DISCOUNT promo codes (i.e. free items or shipping) please enter them during check out in the Notes/Promo Code field. To enter a DISCOUNT promo code enter it into the corresponding box in the 'Promo/% Off' column and then click 'Update' for the given product. If the code is valid for that product the discount percentage will display in place of the box and the amount will become the discounted total for that product(s). If the code is not valid it will remain in the box, please double check the expiry and spelling, and if you feel the code should still be accepted please contact us.
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Data Sheet
Print Version
Organism Human
SourceOrgan Liver
BioSafety Level II
Growth Properties Adherent
Morphology Epithelial
Recommended Seeding Density 10,000 cells/cm2
Population Doubling 72-100 hours
Propagation Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels with the following conditions. Use the ready-to-use Hepatocyte Thawing Medium (TM102) and the Enhanced Primary Human Hepatocytes Media Kit (TM103) which comes with the Hepatocyte Growth Basal Medium, and Hepatocyte Growth Supplement Mix available from abm . Atmosphere: air: 95%, CO₂: 5%; Temperature: 37.0°C.

To make complete Hepatocyte Growth Basal Medium add the entire content of Hepatocyte Growth Supplement Mix into Hepatocyte Growth Basal Medium and mix properly in BSC. Addition of the Hepatocyte Growth Supplement Mix may change medium appearance more opaque.

Procedure Overview
Freeze Thaw Recovery To thaw:

1. Pre-warm Hepatocyte Thawing Medium and fully supplemented Hepatocyte Growth Medium to room temperature.

2. Carefully remove cryovial from storage tank.

3. Thaw cells in 37°C water bath until only a small chunk of ice is left. Do not shake the vial, or take it out of the water during thawing, as this will damage the cells.

4. Spray the vial and the tube containing 50 ml of thawing medium with 70% ethanol and transfer to a Biosafety cabinet.

5. Transfer the now completely thawed cell suspension from the cryovial into 50 ml Hepatocyte Thawing Medium by gently pouring the cells into the medium.

6. Use a 1 ml pipette to transfer 1 ml of the thawing medium back to the cryovial and pour the contents back into the 50 ml tube. Repeat this process twice to completely remove the cells from the cryovial.

7. Pellet the cells by centrifuging at 90×g for 5 min at RT.
Important note: Higher g-forces will significantly reduce cell recovery.

8. Aspirate the supernatant without disturbing the pellet. Leave approximately 200-400 μl medium on top of the cells. 9. Gently loosen and re-suspend the cells without adding any extra medium by agitating and rotating the tube. Do not vortex or shake the cells as this will compromise cell survival.

10. Add an appropriate volume of pre-warmed supplemented Hepatocyte Growth Medium to the pellet (~1ml per million cells thawed) and re-suspend the cells. Avoid pipetting the cells up and down.

11. Determine viable cell number by cell count.

12. Dilute hepatocytes in pre-warmed, fully supplemented Hepatocyte Growth Medium and seed at ~10,000 cells/cm2 in collagen coated cell culture flasks (e.g. T175) or appropriate cell culture dishes.

13. Incubate the cells at 95% humidity, 37°C and 5% CO2.


Note: Where applicable, we recommend selection drug addition after cells recovered from thawing. Directly adding selection drug while thawing could lead to stressed cells and lower viability.
Subculturing To Subculture:

1. Pre-warm PBS, trypsin/EDTA and Hepatocyte Medium to 37°C.

2. Carefully aspirate the culture supernatant.

3. Wash the plate once with ~100 µl PBS/cm2.

4. Add ~50 µl/cm2 trypsin/EDTA (0.05%/0.02% EDTA).

5. Incubate for 3-4 min at 37°C until most of the cells are rounded up and detached. Avoid incubating the cells for more than to 10 min.

6. Gently tap the cell culture vessel to detach remaining adherent cells.

7. Stop the trypsin activity by adding twice the volume (100 µl/cm2) of supplemented Hepatocyte Growth Medium containing 10% FBS (TM999) or Trypsin Neutralization Solution (Lonza).

8. Rinse the surface with the cell suspension using a pipette.

9. Transfer the complete suspension to a tube and centrifuge at 90xg for 5 min at RT.

10. Aspirate the supernatant without disturbing the pellet. Leave approximately 200-400 μl medium on top of the cells. 11. Gently loosen and re-suspend the cells without adding any extra medium by agitating and rotating the tube. Do not vortex or shake the cells as this will compromise cell survival.

12. Add an appropriate volume of pre-warmed supplemented Hepatocyte Growth Medium to the pellet (~1ml per million cells thawed) and re-suspend the cells. Avoid pipetting the cells up and down.

13. Determine viable cell number by cell count.

14. Dilute hepatocytes in pre-warmed, fully supplemented Hepatocyte Growth Medium and seed at ~10,000 cells/cm2 in collagen coated cell culture flasks (e.g. T175) or appropriate cell culture dishes. We strongly recommend end users to purchase the Applied Cell Extracellular Matrix (G422) to coat your cell culture vessels.

15. Incubate the cells at 95% humidity, 37°C and 5% CO2.
Disclaimer 1. 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. All sales are final.
2. We strongly recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping).
3. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.
4. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.
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