HepatoXcell™ Plus: Primary Human Hepatocytes

Unpacking and storage instructions

• Check all containers for leakage or breakage.
• Remove the frozen cells from the liquid nitrogen dry shipper and immediately place the cells at a nitrogen vapor dewar with a temperature below -130°C, until ready for use.

Required media and supplement
• One bottle of each of the following: Hepatocyte Thaw Media (ATCC PCS-450-032), Hepatocyte Plating Media (ATCC PCS-450-038), Hepatocyte Maintenance Media (ATCC PCS-450-034). Cell Basement Membrane (ATCC ACS-3035). Optional media supplements: Penicillin-Streptomycin-Amphotericin B Solution (ATCC PCS-999-002)

Handling procedure
1. Refer to the batch specific information for the total number of viable cells recovered post-thaw for any lot of PCS-450-010.
2. Add 19 mL of pre-warmed Hepatocyte Thaw Media to a 50 mL centrifuge tube.
3. Remove one vial of PCS-450-010 from storage and thaw the cells by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 1 to 2 minutes).
4. Remove the vial from the water bath as soon as the contents are thawed leaving a small ice pellet and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point onward should be carried out under strict aseptic conditions.
5. Gently pour the contents of the vial into the 50 mL centrifuge tube.
6. Using a wide bore pipette tip wash the vial with 1 mL of the Hepatocyte Thaw Media suspension to retrieve any cells left in the vial.
Note: When pipetting Hepatocytes NEVER pipette up and down to mix, instead gently rock or shake the tube.
7. Centrifuge at 100 x g for 10 minutes.
8. Carefully remove the supernatant and resuspend with 1 mL of prewarmed Hepatocyte Plating Media.
9. Gently rock or shake the tube to resuspend the cells, then add an additional 1 mL of Hepatocyte Plating Media.
10. Mix gently by rocking or shaking to ensure a homogenous suspension.
11. Perform a cell count using the Trypan Blue Exclusion Method.
12. Using the total number of viable cells, dilute the cells to 800,000 cells/mL.
13. Seed one well in a Collagen I coated 24-well plate with 500 µL of the cell suspension.
14. Gently shake plate in a “T” motion to evenly distribute the cells.
15. Under the microscope, let the cells settle to ensure seeding density is not too low or high.
16. If the seeding density is too low add 50 µL of cell suspension to the well, if too high shake the plate to resuspend cells and remove 50 µL of cell suspension.
17. After seeding density is optimized, seed remaining wells with Human Hepatocyte cell suspension.
18. Gently shake in a “T” motion and place into a 36°C ± 2°C, 5% CO2 ± 2% CO2 incubator.
19. At 1 hour post seeding gently shake the plate in a “T” motion to evenly distribute the cells and remove any dead cells attached to the plate wells.
20. At 2 hours post-seeding gently shake the plate in a “T” motion and perform a media change using pre-warmed plating media to remove excess dead cells.
21. Four to six hours post-seeding, perform a media change with cold Hepatocyte Maintenance Media supplemented with 0.3 mg/mL Cell Basement Matrix.
22. Put plate back into incubator and change media daily.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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