I am trying to develop embryonic stem cell lines from zebrafish,however I am unsucessful in its development.The literature says High concentration of FBS to be added,however when I am adding higher concentration,a single cell didn't adhere..The cells started to grow at low %ge of serum,however the cells couldnot survive after 2 days of culture,and Fungus starts to grow.Please give sugeestion how I can grow my stem cells for longer period without nay fungal contamination.
Hi,
In my opinion you have a problem with medium composition. Try to culture Zebrafish embryonic stem cells after isolation in one of the two alternative media.
Medium I:
DMEM:F12 Medium
Fetal bovine Serum to a final concentration of 10%.
Antibiotic Antimycotic Solution (100×) 1:100 (A5955, Sigma Aldrich)
or
Medium II:
50% Leibovitz's L-15 medium (ATCC 30-2008)
35% Dulbecco's Modified Eagle's medium, high glucose (GIBCO 12100)
15% F12 medium (GIBCO 21700)
Supplemented with:
0.18 g/L sodium bicarbonate
15 mM HEPES
10% heat-inactivated fetal bovine serum
Antibiotic Antimycotic Solution (100×) 1:100 (A5955, Sigma Aldrich)
Filter the medium.
Add ROCK inhibitor in to the Zebrafish embryonic stem cells up to 24 hours after isolation to enhance cells viability.
Good luck!
I've addaed the antifungal Solution also..but still it starts to grow after 48hrs of incubation..Thank you for your response.
Hi,
Try to wash zebrafish embryo before isolation embryonic stem cells in PBS with Antibiotic Antimycotic Solution (1:100).
After adding FBS and antibiotic to the medium you must filter medium.
Good luck!
Thank you Justyna Augustyniak for your response.I tried the composition of 2nd medium as told by you,n it worked very fine..Thank you for your guidance,now my cells are growing happily without any contamination.
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