Is there any special media I should use for cells that I isolate directly from rat livers? Could I use DMEM or F-12 or do I need something specific to keep these cells alive?
Williams’ Medium E, 5% FCS (for 24 h), 100 nM insulin, 0.05 uM glucagon, 0.1 mg/ml gentamicin, 30 nM Na2SeO3, and 0.1 uM dexamethasone
DMEM with 10% New borne Calf serum. Additives are 10% essential amino acids, 10% non essential amino acids, 10% antibiotic/antimycotic and glutamine.
In Michalopoulos lab, we made our own serum-FREE "HGM" (hepatocyte growth medium). Here is the reference:
Block GD, Locker J, Bowen WC, Petersen BE, Katyal S, Strom SC, Riley T, et al. Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium. J Cell Biol 1996;132:1133-1149.
It is based on based on DMEM. We literally made liters of this stuff!!
How about the RPMI 1640?
We'll coculture the hepatocytes and cancer cells. Our cancer cell is cultured in the RPMI 1640.
Do not know about rpmi, look at its composition versus williamsE commonly used as the DMEM or the mixture 199/DMEM 50/50. Serum is needed only during attachment phase, insulin and glucocorticoids are required for maintaining the phenotype
After 2-step collagenase isolation, we first let the cells attach to Primaria plates for 3 hrs in Hepatocyte attachment medium (DMEM + Pen/strep +10% FCS + 135 nM Insulin +50 nM Dexamethasone). After this step, culture in Hepatozyme +Pen/strep/fungizone
We use williams E' medium consistently in our lab, for fresh hepatocytes isloation.;definitely along with Pen-strep, 5 % FBS, glutamine, dexamethasone and insulin
We also use Williams E' medium. For hepatocyte seeding, the medium is supplemented with 6% FBS, glutamine, dexamethasone, glucagon and insulin. For further cultivation, the medium does not contain FBS.
D-MEM plus with 5% FCS, essential amino acids and antibiotics+antifunginal drugs
I use a culture medium consisting
of MEM/M199 (3 :1) plus 1 g/L BSA, 2.2 g/L NaHCO3,
5 mg/L insulin, 133 IU/L penicillin, 0.1 mg/L streptomycin,
133 IU/L kanamycin and 10% fetal bovine serum
(FBS). In the first 4 h after seeding, the medium also
include 50 μM prednisolone 21-hemisuccinate. Hepatocytes
are seeded at a density of 105 cells/cm2 in 12-well
plastic dishes and incubated at 37 °C under 5% CO2
atmosphere. The medium must be changed every 24 h, and they are OK for at least 96 h. (J Gene Med 2006; 8: 306–313.)
If the isolation of cells is performed under aseptic conditions, using sterile buffers, surgery instruments, etc. there is no need to include antibiotics or antifungal agents.
Williams’ Medium E, 5% FCS (for 24 h), 100 nM insulin, 0.05 uM glucagon, 0.1 mg/ml gentamicin, 30 nM Na2SeO3, and 0.1 uM dexamethasone
William’s
medium E (Life Technologies) supplemented with 1% GlutaMAX I (Life Technologies),
100 IU/mL penicillin, 100 mg/mL streptomycin (Sigma), 6.25 mg/mL insulin,
6.25 mg/mL transferrin, 6.25 ng/mL selenous acid (ITS þ premix; BD Biosciences, San
Jose, CA), 10 mM HEPES (Life Technologies), 50 ng/mL epidermal growth factor (EGF;
PeproTech, Rocky Hill, NJ), 20 ng/mL Gly-His-Lys acetate salt (Sigma), 0.06 nL/mL
ethanolamine (Sigma), 0.01 IU/mL prolactin (Sigma), 1 mg/mL glucagon (Sigma),
2.45 mg/mL cAMP (Sigma), and 40 ng/mL dexamethasone (Sigma).
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