|Species||6-well with |
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.
- 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
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.
|Medium||50 mL||100 mL||250 mL||500 mL|
|Dexamethasone||0.5 µL||1.0 µL||2.5 µL||5.0 µL|
|Maintenance cocktail||2 mL||4 mL||10 mL||20 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 add||2.0 mL||1.0 mL||500 µL||250 µL||125 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.
For questions related to this protocol, contact us at:
Phone: +1 919 237 4500 (Toll)
Phone: +1 866 952 3559 (U.S. Toll-free)