Enhanced Primary Human Hepatocytes - 5 million

Cat. No.
T5996
Unit
5x105 cells / 1.0 ml
Price
Inquiry
Cat. No. T5996
Name Enhanced Primary Human Hepatocytes - 5 million
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.
Organism Human (H. sapiens)
Tissue Liver
Growth Properties Adherent, polygonal
Cell Type Primary Cells
Unit 5x105 cells / 1.0 ml
Storage Condition Vapor phase of liquid nitrogen, or below -130°C.
Shipping Conditions Ship with dry ice.
Product Format Frozen
Intended Use This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
BioSafety II
Certificate of Analysis For batch-specific test results, refer to the applicable certificate of analysis that can be found at www.abmgood.com.
Growth Conditions

PriCoat™ ECM T25 Flasks (G999) or Applied Cell Extracellular Matrix (G422) are required for cell adhesion to the culture vessels.  Ready-to-use Hepatocyte Thawing Medium (TM102) and the Hepatocyte Growth Medium Kit (TM103) which comes with the Hepatocyte Growth Basal Medium, and Hepatocyte Growth Supplement Mix are required to grow the cells, 37.0°C, 5% CO₂.
Unpacking and Storage Instructions

1. Visually examine the packaging containers for signs of leakage or breakage.

2. Immediately transfer frozen cells from dry ice packaging to a temperature below -130°C, preferably in liquid nitrogen vapor phase storage, until ready for use.

To ensure the highest level of viability, thaw the vial and initiate culture as soon as possible upon receipt. If continued storage is desired, the vial should only be stored below -130°C or in liquid nitrogen vapor phase. Do not store at -70°C, as it will result in loss of viability.
Thawing Protocol

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 (~1 ml 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 ECM coated cell culture flasks (e.g. T175) or appropriate cell culture dishes.

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

14. Change media the next day.


Note: Cells can be maintained for up to 18 days post-plating. For extended culture, replace the medium every 2–3 days to support optimal cell health and function.
Subculture Protocol

Volumes given below are for a T75 flask; proportionally increase or decrease the volume as required per culture vessel size. Subculture cells once the culture vessel is 70-80% confluent.

1. Aspirate the culture media, and wash cells with 100 μl PBS/cm2. Add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel.

2. Observe the cells under a microscope to confirm detachment (typically within 3-4 minutes). Cells that are difficult to detach can be put in 37°C, for several minutes to facilitate detachment. Gently tap the cell culture vessel to detach remaining adherent cells. Do not incubate the cells for more than 7 minutes.

3. Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media with 10% FBS into the culture vessel.

4. Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 90xg for 5 minutes. 

5. Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new coated culture vessels, as desired.

6. Incubate the cells at the recommended conditions.
Cryopreservation

We do not recommend to freeze down the Enhanced Primary Human Hepatocytes.
Seeding Density (cells/cm2) 10,000
Warranty abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period”.
Disclaimer

1. 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 licensing@abmgood.com.

2. 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 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final.

3. We 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).

4. 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.

5. 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.

6. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period."
QC

Each lot has been tested for:

  1. >90% plateability

  2. Post-thaw viability

  3. CMV, HIV, HBV, HCV, and Mycoplasma negative

  4. Hepatic markers via immunofluorescence staining: CK8+, AAT+, CK18+, HAS+, and AFP-

  5. Basal and inducible CYP activities

  6. Glycogen storage via PAS staining
Application Research Use Only.
Material Citation If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. T5996
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