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Primary Human Suspension Hepatocytes Culture Protocol

Primary Human Suspension Hepatocytes

Our extensive collection of normal human primary liver cells includes characterized suspension Hepatocytes, which can be used to model the function of Hepatocytes in vitro. Suspension cells in culture float in media and don’t form a monolayer on the culture surface. We currently offer two options for the characterized suspension Hepatocytes: the mixed gender pool and the single donor. Our pooled suspension Hepatocytes are normalized for cell number to ensure that each donor contributes an equal contribution to the pool. Our single donor human suspension consists of a homogenous population of Hepatocytes and it is offered in various pack sizes. These primary suspension Hepatocytes are ideal for the studies of enzyme induction, toxicity, drug screening, transporter efflux activity, and potential drug-drug interactions.

Our primary Hepatocytes collection are isolated from whole human livers, with each donor providing documented consent for research use of non-transplantable organs or tissues. The suspension Hepatocytes cells are cryopreserved for ease of use and undergo testing for enzyme profiling data and ensuring a post-thaw viability of ≥70%.

In this protocol, we demonstrate how to thaw and grow primary Hepatocytes in suspension culture.

Primary Human Suspension Hepatocytes Culture Materials

  • Human Characterized Suspension Hepatocytes 20-Donor Mixed Gender Pool (HLP120-5M) or 10-Donor Mixed Gender Pool (HLP110-5M) or Normal Human Characterized Suspension Hepatocytes (HLP101). Note: Upon receipt, immediately store cryovial(s) in liquid nitrogen.
  • 50ml Centrifuge Tube
  • Tissue culture treated multiwell plates
  • 1X Hepatocyte Plating Medium (HPM) for Human Hepatocytes

1X Maintenance Medium for Human Hepatocytes

Note: For Dexamethasone, dissolve 25 mg into 32 mL of ethanol (100%) to make 2mM stock. For Linoleic acid, dissolve 100mg into 2mL of ethanol (100%) to make 50mg/mL stock.

Suspension Hepatocytes Growth Protocol

The protocol was performed within a Class II laminar flow biohood unless otherwise specified. Incubators were set to 37oC and 5% CO2. PPE such as safety glasses, gloves, and lab coat should be worn.

Thawing and Plating Suspension Hepatocytes

  1. Fill a 50mL centrifuge tube with 45mL of 4oC Hepatocyte Plating Medium (HPM).
  2. Remove hepatocyte vial(s) from storage tank or freezer.
  3. Immerse the vial up to the cap into the 37oC water bath. Be careful not to completely submerge the cap.
  4. Gently shake the vial and shake until the ice pellet has melted to the point of a small spindle.
    Note: Do not fully thaw the cell suspension. It will take approximately 90 – 120 seconds.
  5. Remove the vial from the water bath. Spray with 70% IPA or wipe down with a 70% IPA wipe and transfer into the bio safety cabinet.
  6. Pour the contents of the vial into the 50mL conical containing HPM.
  7. Remove 1mL of this medium and hepatocyte suspension using a pipette and place into the vial, ensuring collection of any remaining cells. Place in conical.
  8. Invert the conical gently 3 to 4 times to ensure resuspension of the hepatocytes.
  9. Centrifuge the conical at 100xg for 10 minutes.
  10. Aspirate the supernatant.
  11. Resuspend the cell pellet in 3-4mL of 37oC Hepatocyte Maintenance media. Gently resuspend the pellet by slowly rocking the 50ml conical back and forth, allowing the media to repeatedly wash over the pellet until fully resuspended.
  12. Calculate the total cell count and cell viability using the Trypan Blue Exclusion method:

    a. Prepare a 1:10 dilution by combining 400µL of media with 50µL of Trypan Blue, then add 50µL of well-mixed cell suspension.

    b. 1:5 = 350µL media + 50µL trypan + 100µL of cell suspension.

    c. 1:2 = 200µL media + 50L µof trypan + 250µL of cell suspension.

    i. Calculate cell viability percentage: {Live Cells/(Live + Dead Cells)}*100
    ii. Calculate cell yield per mL: (Total Live Cells/Number of Squares Counted)*10,000*Dilution Factor

  13. Add additional Hepatocyte Maintenance Medium to reach the desired cell concentration for experimental design.

Primary Suspension Hepatocyte Growth Results

The primary hepatocytes grown in suspension (pooled and non-pooled samples) were tested for viability using the Phase I (CYP) and II (UGT, SULT) metabolism activity via ECOD assay, which measures the rate of clearance of 7-ethoxycoumarin and the rate of formation of metabolites 7-hydroxycoumarin sulfate (7-HCS) and 7-hydroxycoumarin glucuronide (7-HCG). Hepatocytes in suspension were incubated for 60 min at 37oC under 5% CO2 and saturated humidity with substrates as indicated the table shown below; samples were terminated with 3x acetonitrile containing the analytical internal standard. Metabolites were analyzed by LC-MS/MS.

Table 1. Example of donor demographics of 20 pooled hepatocytes for primary pooled suspension hepatocytes.
Table 2. Example results of Phase I (CYP) and II (UGT, SULT) metabolism activity for pooled suspension hepatocytes.
Table 3. Example results of Phase I (CYP) and II (UGT, SULT) metabolism activity via ECOD assay for primary suspension hepatocytes.
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