|Description||Enhanced Primary Human Hepatocytes are expanded primary cells which retains the physiologically relevant profile and phenotype of primary hepatocytes. These cells are ready-to-use for applications in research and development, such as viral infections, screening, co-cultures, transient transfection, and for metabolism studies. The late expansion cells are tested for CYP induction and inhibition. It is not recommended for further extensive passaging after thawing but can be used for long term culture which is necessary for cell-based assays.
Enhanced Primary Human Hepatocytes -1 Million is a lower cell count of our expanded primary hepatocytes offered at abm. We have a higher cell count available as the Enhanced Primary Human Hepatocytes – 5 Million (Cat. T5996).
|Species||Human (H. sapiens)|
|Applications||For Research Use Only|
|Seeding Density||10,000 cells/cm2|
|Donor Gender||Donor Info Not Disclosed|
|Cell Type||Primary Cells|
|Propagation Requirements||Use of 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. We strongly recommend end users to purchase the Applied Cell Extracellular Matrix (G422) to coat your cell culture vessels. 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%, CO?: 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 BioSafety Cabinet. Addition of the Hepatocyte Growth Supplement Mix may change medium appearance more opaque.
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.
8. Aspirate the supernatant without disturbing the pellet. Leave approximately 200-400 ?l 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.
|Subculture Protocol||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.
|Preservation Protocol||We do not recommend freezing down the Enhanced Primary Human Hepatocytes.|
|Unit quantity||1×106 cells / 1.0ml|
|QC||Each lot has been tested for:
|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. 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 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.
What is the supply procedure?
The frozen vial of cells will be shipped in Dry ice while T25 flasks by themselves are shipped at room temperature.
Does ''Normal" mean undiseased tissue/organ?
How often do I need to change the media?
The media should be renewed every 2-3 days
How should I store frozen vials of primary cells? Can a frozen vial be put back into liquid nitrogen after delivery?
In most cases, a vial of primary cells shipped in dry ice (-70°C) can be placed back into liquid nitrogen and recovered at a later date by rapid thawing. However it is important to note that the viability of some sensitive cell types may be reduced by the temperature shift of such treatment, making recovery more difficult. For this reason, we recommend that cells be thawed and placed into culture as soon after receipt as possible. It is best to minimize storage time at -70°C; as this is only used for shipping the cells. ABM does not warrant the viability of cells stored at -70C after shipments have been received.
How much Pen/Strep should I add to the culture media? (Only where stated in the culturing protocol provided).
We suggest 1% P/S (G255). It should not be required if your lab does not use P/S routinely however. G255 Penicillin-Streptomycin contains 10,000 units of penicillin (base), 10,000µg of streptomycin (base) per mL in WFI water. It is supplied as a 0.22 µm-filtered, 100X frozen liquid.
What is the recommended storage temperature?
In general, if you received: Live cells: acclimatize for 3-4 hrs at 37C, 5% CO2 and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen or dry ice; -180C.
Is it possible to freeze the cells again after thawing?
Unless specified otherwise in the datasheet, primary cell lines may be re-freezed after thawing. However, primary cells are usually not good for multiple passaging and they usually last only for up to 10-12 population doublings. Our primary cells are usually provided at passage 2, therefore they can be passaged for an additional 1-2 passages. For long term use, immortalized cells are preferred.
Can I seed frozen cells directly into collagen coated 6-well plates? Or I should seed in T25 flasks (G299) first, and then transfer to 6-well plate?
If you wish to use a 6-well plate, you can seed it directly into the plate; no need to go through T25 flask then to a 6-well plate.
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. Seeding density is important as many 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.
How many cells are each vial?
The number of cells in a vial is lot-dependent. A Certificate of Analysis stating the cell quantity of the vial will be provided with your order.