實驗概要
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.
實驗步驟
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).