Introduction

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.

Materials

Recommended reagents Size Cat. No.

Human suspension

   
Thawing Medium
  • Cryopreserved Hepatocytes Recovery Medium (CHRM®)
50 mLCM7000
Incubation Medium
  • Williams’ Medium E (1x, no phenol red) 
  • Hepatocyte Maintenance Supplement Pack (Serum-free)

500 mL
1 pack


A1217601

CM4000

 

Recommended reagents  Size  Cat. No.

Human plateable

   
Thawing Medium
  • Cryopreserved Hepatocytes Recovery Medium (CHRM®)
50 mLCM7000
Plating Medium
  • Williams’ Medium E (1x, no phenol red) 
  • Hepatocyte Plating Supplement Pack (Serum-containing)

500 mL
1 pack

A1217601
CM3000   
Incubation Medium
  • Williams’ Medium E (1x, no phenol red)
  • Hepatocyte Maintenance Supplement Pack (Serum-free)
  • Collagen I, Coated Plates 
  • Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix

500 mL
1 pack
1 plate
5 mL


A1217601

CM4000

CM1024

A1413201

 

Recommended reagents Size Cat. No.

Animal suspension

   
Thawing Medium
  • Williams’ Medium E (1x, no phenol red)
  • Hepatocyte Plating Supplement Pack (Serum-containing)

500 mL
1 pack


A1217601

CM3000

Incubation Medium
  • Williams’ Medium E (1x, no phenol red) 
  • Hepatocyte Maintenance Supplement Pack (Serum-free)

500 mL
1 pack


A1217601
CM4000

 

Recommended reagents Size  Cat. No. 

Animal plateable

   
Thawing Medium
  • Williams’ Medium E (1x, no phenol red)  
  • Hepatocyte Plating Supplement Pack (Serum-containing)

500 mL
1 pack

A1217601
CM3000
Plating Medium
  • Williams’ Medium E (1x, no phenol red) 
  • Hepatocyte Plating Supplement Pack (Serum-containing)

500 mL
1 pack

A1217601
CM3000   
Incubation Medium
  • Williams’ Medium E (1x, no phenol red)
  • Hepatocyte Maintenance Supplement Pack (Serum-free)
  • Collagen I, Coated Plates
  • Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix

500 mL
1 pack
1 plate
1 mL

A1217601
CM4000
CM1024
12760021



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

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Ordering Information

Catalog # Name Size List Price (USD) Qty
HMCPIS Human Plateable Hepatocytes, Induction Qualified 1.5 mL 1,314.00
RTCS10 Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Male 1.5 mL 191.00
RTCS20 Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Female 1.5 mL 191.00
RTCP10 Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Plateable Male 1.5 mL 387.00
RTCP20 Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Plateable Female 1.5 mL 387.00
MKCS10 Monkey (cynomolgus) Cryopreserved Hepatocytes, Male 1.5 mL 363.00
MKCS20 Monkey (cynomolgus) Cryopreserved Hepatocytes, Female 1.5 mL 363.00
MKCP10 Monkey (cynomolgus) Cryopreserved Hepatocytes, Plateable Male 1.5 mL 720.00
MKCP20 Monkey (cynomolgus) Cryopreserved Hepatocytes, Plateable Female 1.5 mL 720.00
MSCS10 Mouse (CD-1) Cryopreserved Hepatocytes, Male 1.5 mL 520.00
MSCS20 Mouse (CD-1) Cryopreserved Hepatocytes, Female 1.5 mL 520.00
MSCP10 Mouse (CD-1) Cryopreserved Hepatocytes, Plateable Male 1.5 mL 980.00
MSCP20 Mouse (CD-1) Cryopreserved Hepatocytes, Plateable Female 1.5 mL 980.00
DGCS10 Dog (Beagle) Cryopreserved Hepatocytes, Male 1.5 mL 273.00
DGCS20 Dog (Beagle) Cryopreserved Hepatocytes, Female 1.5 mL 273.00
DGCP10 Dog (Beagle) Cryopreserved Hepatocytes, plateable Male 1.5 mL 564.00
DGCP20 Dog (Beagle) Cryopreserved Hepatocytes, plateable Female 1.5 mL 564.00
HMCPMS Human Plateable Hepatocytes, Metabolism Qualified 1.5 mL 1,026.00
HMCPTS Human Plateable Hepatocytes, Transporter Qualified 1.5 mL 1,520.00
HMCSPS Human Suspension Hepatocytes, Polymorphic Donors, (4-8 x 106 cells) 1 vial 788.00
HMCSPL Human Suspension Hepatocytes, Polymorphic Donors, (9-12 x 106 cells) 1 vial 994.00
HMCPUS Human Plateable Hepatocytes, Uptake Qualified 1.5 mL 1,026.00
HMCS1S Human Suspension Hepatocytes, Metabolism Qualified, Male, (4-8 x 106 cells) 1 vial 479.00
HMCS1L Human Suspension Hepatocytes, Metabolism Qualified, Male, (9-12 x 106 cells) 1 vial 640.00
HMCS2S Human Suspension Hepatocytes, Metabolism Qualified, Female, (4-8 x 106 cells) 1 vial 479.00
HMCS2L Human Suspension Hepatocytes, Metabolism Qualified, Female, (9-12 x 106 cells) 1 vial 640.00
HMCSTS Human Suspension Hepatocytes, Transporter Qualified, (4-8 x 106 cells) 1 vial 670.00
HMCSTL Human Suspension Hepatocytes, Transporter Qualified, (9-12 x 106 cells) 1 vial 876.00
HMCS10 HEP10, Pooled Human Cryopreserved Hepatocytes 1.5 mL 800.00

Protocol

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)
     
  1. Thaw cryopreserved hepatocytes in 37°C water bath for <2 min.
  2. 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).
  3. 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.
  1. 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

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

  3. 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
  1. 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.
  2. 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.
  1. After incubation, agitate plates to loosen debris and aspirate medium.
  2. 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.

 

  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.
  1. Find the protein concentration of Geltrex™ Matrix on its specification sheet - each lot is slightly different.
  2. 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
  1. Add Geltrex™ Matrix to the cold incubation medium on ice.  Mix well by pipeting several times.
  2. 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.
  1. Replace incubation medium daily.


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

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Tables

Table 1—General Seeding Density Guide for Cyropreserved Hepatocytes. 12mL media per plate. Note: each lot may require slight adjustments in seeding density to form optimal monolayer. Please contact Technical Support with any questions – 1-866-952-3559.

Species 6-well 12-well 24-well 48-well 96-well
Human0.9-1.1 x 106 cells/mL0.8-1.0 x 106 cells/mL0.7-0.9 x 106 cells/mL0.6-0.8 x 106 cells/mL0.5-0.7 x 106 cells/mL
Rat0.9-1.1 x 106 cells/mL0.8-1.0 x 106 cells/mL0.7-0.9 x 106 cells/mL0.6-0.8 x 106 cells/mL0.5-0.7 x 106 cells/mL
Dog0.9-1.1 x 106 cells/mL0.8-1.0 x 106 cells/mL0.7-0.9 x 106 cells/mL0.6-0.8 x 106 cells/mL0.5-0.7 x 106 cells/mL
Monkey1.1-1.3 x 106 cells/mL1.0-1.2 x 106 cells/mL0.9-1.1 x 106 cells/mL0.8-1.0 x 106 cells /mL0.7-0.9x 106 cells/mL
Mouse0.5-0.7 x 106 cells/mL0.4-0.6 x 106 cells/mL0.3-0.5 x 106 cells/mL0.2-0.4 x 106 cells/mL0.1-0.3 x 106 cells/mL


Table 2—Approximate Number of Cells Per Plate . 12mL media per plates.

Species 6-well 12-well 24-well 48-well 96-well
Human10.8-13.2 million cells9.6-12 million cells8.4-10.8 million cells7.2-9.6 million cells6-8.4 million cells
Rat10.8-13.2 million cells9.6-12 million cells8.4-10.8 million cells7.2-9.6 million cells6-8.4 million cells
Dog10.8-13.2 million cells9.6-12 million cells8.4-10.8 million cells7.2-9.6 million cells6-8.4 million cells
Monkey13.2-15.6 million cells12-14.4 million cells10.8-13.2 million cells9.6-12 million cells8.4-10.8 million cells
Mouse6-8.4 million cells4.8-7.2 million cells3.6-6 million cells2.4-4.8 million cells1.2-3.6 million cells
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Technical support

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.

LT130       Rev.  28-Feb-2011