Thawing and Plating Cryopreserved Hepatocytes

This protocol covers the thawing and prep of cryopreserved hepatocytes for their subsequent use in applications such as metabolic stability (intrinsic clearance), metabolite ID/profiling, enzyme induction, hepatotoxicity, transporter uptake and efflux, environmental bioaccumulation and liver disease research. The initial part of this protocol is suitable for suspension lots; follow the entire protocol for plating and overlay of plateable hepatocytes.

* (See www.lifetechnologies.com/hepatocytes for alternative sizes and options)

Advance preparation

• If plating hepatocytes with an overlay, refer t o the specification sheet for Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix’s (cat. no. A1413201), which will provide the lot’s concentration and technical tips. Geltrex™ Matrix should be thawed on ice for 2—3 hours prior to application, or overnight at 4ºC, and kept ice-cold to prevent gelling.
• Read the directions supplied with the Hepatocyte Maintenance and Plating Supplement Packs (CM4000 and CM3000) and prepare Maintenance and Plating Media using Williams’ Medium E.
• Review this protocol to ensure you have all the necessary reagents and equipment prior to starting the procedure. Once thawed, cryopreserved hepatocytes must be used immediately and will not retain metabolic activities if re-frozen.
• Not all cryopreserved hepatocytes are suitable for plating. If using this protocol for plating hepatocytes, confirm that the lot is plateable.

(Use universal safety precautions and appropriate biosafety cabinet when handing primary hepatocytes.)


Thaw, spin, resuspend

1. Warm these media to 37°C in a water bath:

• CHRM® Medium (for human) or ~ 48 mL of Thawing Medium: Williams Medium E and Hepatocyte Plating Supplement Pack (for animal)
  Plating medium (if plating hepatocytes; this is Williams Medium E supplemented with Hepatocyte Plating Supplement Pack, Serum-Containing)
• Incubation medium (for suspension only this is Williams Medium E supplemented with Hepatocyte Maintenance Supplement Pack, Serum-free)

2. Thaw cryopreserved hepatocytes in 37°C water bath for <2 min.
3. Wipe the vial with 70% alcohol in hood; pour or use wide-bore pipette tip to transfer hepatocytes into CHRM® Medium (for human) or Williams Medium E and Hepatocyte Plating Supplement Pack (for animal).
4. Centrifuge at room temperature:

• Human hepatocytes, 100 x g for 10 min.
• Dog and non-human primate hepatocytes, 65 x g for 4 min.
• Rat and mouse hepatocytes, 55 x g for 3 min.

5. Pour supernatant off into waste bottle and invert completely. Do not shake. Add ~1 ml of the following per 1 x 10⁶ total cells:

• Plating medium, if plating the hepatocytes,
• or pre-warmed Incubation Medium, if using cells in suspension.

Count, plate, and incubate

6. Determine cell viability and yield.  For help, see:

• Cell counting calculation worksheet
• Note:  Hepatocytes can be slightly problematic in automated cell counting instruments.  We suggest manual counting for better accuracy, as the correct plating density is critical for good results.

7. If using the hepatocytes in suspension, add additional medium to bring cells to desired concentration (i.e. 1x10⁶ cells/mL)—do not proceed with the subsequent plating steps.
8. Dilute to correct seeding density with Plating Medium. (Table 1, Table 2 at bottom of page)
   
   
9. Transfer hepatocytes to multi-well plate. For a 24-well plate:

• Human hepatocytes, 500 µL, density according to Product Characterization Sheet
• Dog and rat hepatocytes, 500 µL, 3.5–4.5 x 10⁵ cells per well
• Mouse hepatocytes, 500 µL, 1.5–2.5 x 10⁵ cells per well
• Non-human primate hepatocytes, 500 µL, 4.5–5.5 x 10⁵ cells per well

10. Place plate in incubator, and with hand on top of lid disperse cells with very slow figure-eight and north/south and east/west motions.
11. Incubate plate at 37°C for 4–6 hr.

• Do not move plate during this time, as the cells are forming a monolayer.
• If planning to overlay hepatocytes, use this time to calculate the amount of Geltrex™ Matrix needed.

12. After incubation, agitate plates to loosen debris and aspirate medium.
13. If using an overlay, proceed to the next step.  If not using an overlay, replace medium with warm Incubation Medium, or alternative medium, depending on your application.  Do not let the hepatocytes dry out - replace medium quickly.

Overlay

Important Note

Geltrex™ Matrix and the Incubation Medium used for its dilution must be kept ice cold to prevent premature gelling.  Keep Geltrex™ Matrix and Incubation Medium on ice; preferably use cold pipettes when mixing.

14. Calculate the amount of incubation medium needed to feed the plated hepatocytes and place this volume on ice.

• Generally, this is 12 mL per plate; consider adding 1-2 mL for a slight excess of solution.

15. Find the protein concentration of Geltrex™ Matrix on its specification sheet - each lot is slightly different.
16. Multiply the amount of incubation medium by our recommended final Geltrex™ Matrix concentration of 0.35 mg/mL, and divide by the protein concentration of Geltrex to get the amount of Geltrex™ Matrix that needs to be added to the incubation medium:

• (mL incubation medium x 0.35 mg/mL)/Geltrex™ protein conc. = mL of Geltrex™ to add

17. Add Geltrex™ Matrix to the cold incubation medium on ice.  Mix well by pipeting several times.
18. Apply overlay to plated hepatocytes and incubate at least two hours or up to 24 hr prior to use.

• The gel layer will settle out of the media over the top of the hepatocytes.

19. Replace incubation medium daily.

Note: For overlaying dog hepatocytes we recommend using Matrigel™ (BD) or ECM (Sigma).

For questions related to this protocol, contact us at:
Email: hepaticproducts@lifetech.com
Phone: +1 919 237 4500 (Toll)
Phone: +1 866 952 3559 (U.S. Toll-free)

CHRM® is a registered trademark of APSciences.