Hello all,
I'm trying to isolate and expand human primary renal fibroblasts from nephrectomy samples using an explantation technique. This technique has already been used successfully in my lab for the isolation and propagation of human skin fibroblasts.
At first, the cells properly grew out of the explanted tissue and looked healthy at D14, with a classic fingerprint pattern. However, after the first passaging step, they started to appear morphologically altered (round, irregular in shape, vacuolated) and were very slow to grow.
I am using the same medium that has previously been used for the culture of skin fibroblasts (DMEM + 10% FCS) and of human primary renal fibroblasts (references below). I tried two different batches of FCS, with the same results.
I am using Sarstedt Cell+ tissue culture flasks (yellow cap).
What could be a reason for the issue I am facing?
Thanks in advance.
References:
10.1007/978-1-59745-352-3_3
10.1002/cbin.11150
Hello Stéphane Lang
The primary cells become senescent when grown to 100% confluency. I would recommend sub culturing the cells when they are 85-90% confluent. When the cells overgrow the morphology changes.
Trypsin can be harmful to the primary cells and passaging primary cells too frequently is not recommended. Over trypsinization when sub culturing primary cells should be avoided. Use low concentration of trypsin and monitor the cells under the microscope during the process. Immediately neutralize the cells after trypsinization as active trypsin will damage primary cells.
Use low serum in culture medium in order to reduce the negative effects of growth inhibitory factors on the cells. Instead, you can supplement the growth medium with growth factors, hormones, amino acid mixture to compensate for low serum. If you have used 10% FCS in growth medium before with success then continue using it.
I hope this information will help.
Good Luck.
Hi, I do think your cells are overconfluent. Try to subculture at low confluency. Sometime, do your incubator too for the consistency of CO2.
Your cells are possibly senescent. Is it possible the problem lies with your nephrectomy sample, in that the donor is older? I used to grow mesangial cells from older donors and they would become senescent after a few passages.
Stéphane Lang try using RPMI1650 or MEM with 10%FBS if you keep observing slow growth. DMEM is good for renal cells and your morphology of cells is also looking good, if you still have doubts then you can cross check with morphology of HEK 293 cell line.
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