Thawing and Plating Cryopreserved Hepatocytes

实验概要

This  protocol covers thawing and prep of cryopreserved hepatocytes for  applications such as metabolic stability, intrinsic clearance, 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.

实验步骤

1.        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.

2.        Count, plate, and incubate

1)        Determine cell viability and yield.

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.

2)        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.

3)        Dilute to correct seeding density with Plating Medium. (Table 1, Table 2 at bottom of page)

4)        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

5)         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.

6)        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.

7)        After incubation, agitate plates to loosen debris and aspirate medium.

8)         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.

3.        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.

1)        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.

2)        Find the protein concentration of Geltrex™ Matrix on its specification sheet - each lot is slightly different.

3)         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

4)        Add Geltrex™ Matrix to the cold incubation medium on ice.  Mix well by pipeting several times.

5)        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.

6)        Replace incubation medium daily.

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