Materials

Species 6-well with
Geltrex
6-well
without
Geltrex
12-well
with
Geltrex
12-well
without
Geltrex
24-well
with
Geltrex
24-well
without
Geltrex
48-well
with
Geltrex
48-well
without
Geltrex
96-well
with
Geltrex
96-well
without
Geltrex
Rat RTFY06 RTFN06RTFY12
RTFN12
RTFY24
RTFN24
RTFY48
RTFN48
RTFY96
RTFN96
Mouse MSFY06MSFN06 MSFY12
MSFN12
MSFY24
MSFN24
MSFY48
MSFN48
MSFY96
MSFN96
Dog
DGFY06*
DGFN06
DGFY12*
DGFN12
DGFY24*
DGFN24
DGFY48*
DGFN48
DGFY96*
DGFN96
Monkey
MKFY06
MKFN06
MKFY12
MKFN12
MKFY24
MKFN24
MKFY48
MKFN48
MKFY96
MKFN96
Human
HMFY06
HMFN06
HMFY12
HMFN12
HMFY24
HMFN24
HMFY48
HMFN48
HMFY96
HMFN96

 
Instructions for handling fresh hepatocytes after receipt of shipment:

Important – Please Read!

The shipping medium for GIBCO® Fresh Hepatocytes should be exchanged with new medium, using the following instructions, immediately upon receipt.

Precautions

  • Treat all hepatocytes as potentially hazardous material
  • Wipe surfaces, exteriors of bottles and hepatocyte containers with 70% isopropanol
  • Perform all operations using aseptic handling technique in a sterile environment

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Protocol

Step 1—Prepare Supplemented Medium

Supplement Williams Medium E without Phenol Red (Cat. No. A1217601), or suitable alternative basal medium, with the appropriate amount of Maintenance Supplement Pack, Serum-free (Cat. No. CM4000). See Table 1. Filtering is recommended, but not required. Use a water bath to warm the supplemented medium to 37°C prior to use with hepatocytes.

Contents of Maintenance Supplement Pack, Serum-free:
  • Dexamethasone, 10 mM in DMSO solution, 1 x 65 μL (supplied in excess)
  • Maintenance Cocktail solution, 1 x 20 mL, containing:
  • 2.5 mL penicillin-streptomycin, 10,000 U/mL
  • 5.0 mL ITS+ (insulin, transferrin, selenium complex, BSA, linoleic acid)
  • 5.0 mL GlutaMAX™, 100X
  • 7.5 mL HEPES, pH 7.4; 1M

Table 1—Volumes for supplementing medium with Maintenance Supplement Pack, Serum-free (Cat. No. CM4000) for use of hepatocytes in suspension. Each plate requires 12 mL of supplemented media daily.

Medium50 mL100 mL250 mL500 mL
Dexamethasone0.5 µL1.0 µL2.5 µL5.0 µL
Maintenance cocktail2 mL 4 mL10 mL20 mL*


Step 2—Remove and Replace Medium on Hepatocyte-containing Plates

A. Remove hepatocyte plates from shipping container; record lot number.
B. Place plate(s) in biosafety cabinet and remove all packaging.
C. Carefully aspirate all shipping media from each well.
D. Gently replace with appropriate volume of supplemented cell culture medium. See Table 2.

Table 2: 
Maintenance medium volume per well for various plate formats


  6-well plate 12-well plate 24-well plate 48-well plate 96-well plate
Volume of medium to add2.0 mL1.0 mL500 µL250 µL125 µL

 
Step 3—Incubate and Replace Medium Daily

E. Place plates in a humidified incubator, 5% CO2 at 37°C and allow hepaotcytes to acclimate overnight prior to  use (approximately 18 hours).

F. Feed cells with fresh supplemented culture medium daily. The optimal experimental conditions must be determined by the investigator according to the application.
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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)

LT140              7-Mar-2011