I am encountering a challenge with culturing endothelial cells derived from umbilical cord tissue. Despite successful isolation, the cells exhibit a decline in viability and begin to deteriorate after 3-4 days in culture. I have attempted to address this issue by utilizing various brands of endothelial cell media, yet the problem persists. Enclosed is image depicting the condition of the cell culture flask. Any insights or recommendations regarding this issue would be greatly valued.
Hello Abhinav,
I've noticed that you've been working on isolating endothelial cells, and I'd like to offer some suggestions based on the provided image. From what I see, it appears that the cell density is quite low.
Incorporating these adjustments into your cell culture protocol should help improve cell density and overall culture conditions. If you have any further questions or need additional assistance, feel free to reach out. Best of luck with your experiments!
Warm regards,
ANJALI ROY
Hi. This is how I used to collect HUVEC, and despite now and then we had some problems due to variations related to the donors, in general we had very good load of cells and usually they were easy to work with them for a few (3-6) passages.
"Human umbilical vein endothelial cell (HUVEC) cultures were
obtained by the method of Jaffe et al. (1973). Briefly, the cells were
obtained by flushing umbilical veins obtained from the maternity unit
(City Hospital, Nottingham, UK) with warmed 0.05% collagenase
solution. After collection, the cells were washed and re-suspended in
HUVEC culture medium (40% Medium 199, 40% F12 nutrient mix, 1 M
HEPES, 200 mM NaHCO3, 2 mM L -glutamine, 50 ng/mL bFGF, 20 ng/
mL EGF, 3000 units of heparin, 20% sterile deionized water, and 10% of
FBS) and grown until confluent in T-25 tissue culture flasks coated with
0.2% gelatin." (Article E-Selectin expression in human endothelial cells exposed to ...
,)
Hello Abhinav,
The following might help to resolve this issue:
1). Increase density of passage 1 cells (to at least 30-40% confluency).
2). Pre-coat flask with medium and equilibrate in incubator at 37C for 30-60 min before seeding cells.
3). Check for mycoplasma contamination.
4). Using correct plasticware/flasks is important too.
All the best,
Leonid
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