Dear all,
When I culture the stromal vascular fraction (SVF) from murine inguinal adipose tissue, the size of the cells increase. I see this when passaging my cells the first time and the following passages. I wonder if my cells are differentiated, senescent / dying, or if it is normal for them to be bigger compared to the initial SVF cells.
I have added some pictures
I don’t think it is density related, because I see this phenomenon in all my culture dishes (with cells at a different density).
I also don’t think the cells are already differentiated or senescent, because they expand quite efficiently (cell number x2-x3 every 2 days), I used new stromal medium and the first passage seems a little too early to be already differentiated or senescent / dying. I could try do repeat my experiment with adding bFGF to my medium to delay senescence / differentiation.
I haven’t performed flow cytometry yet to test surface markers in these cells but I am planning to buy the antibodies soon.
When I look at the 3rd picture (at higher magnification), it also doesn’t look like my cells are aggregated or clotted together. I could add heparin to prevent this, but doesn’t look necessary at the moment.
I wonder if someone else with ASC culturing experience has seen this? Is it normal?
All help is appreciated! Thank you (more details are described below)
Details about the source:
- 4 mice, C57BL/6 wild type, 4 months old, female
Details about the procedure:
- inguinal adipose tissue is harvested, after the inguinal lymph nodes are removed
- washing in HBSS (the adipose tissue from the 4 mice was put together in one 50-ml tube)
- mincing to obtain semi-liquid adipose tissue
- digestion in collagenase type 1 (0.12%) at 37°C for 1 hour (vortex every 5 minutes)
- neutralisation of the collagenase activity with stromal medium (DMEM + 10% FBS + 1% penicillin-streptomycin, all compounds are new)
- strain through a 40-µm filter
- centrifugation at 300g for 5 minutes
- aspirate supernatant, add new stromal medium, mix manually and thoroughly for 10 seconds
- centrifugation at 300g for 5 minutes
- aspirate supernatant and resuspend in stromal medium, cell count
Initial SVF yield:
- SVF: 1.42 x 10^7 cells (combined for the 4 donor mice)
Plating:
- all plates have a diameter of 10 cm
- I plated dishes with 5 x 10^5 cells (6 400 cells / cm²), dishes with 1 x 10^6 cells (12 800 cm²) and a dish with 5 x 10^6 cells (64 000 cells / cm²)
- removal of non-adherent cells after 24 hours and medium change every 48 hours
Passaging
- 80% confluency on day 6 in the plate with the initial 5 x 10^6 cells
- 80% confluency on day 8-9 in the other plates (with 5 x 10^5 or 1 x 10^6 cells)
- collection of cells with trypsin (0.25% in EDTA, max. 2 minutes)
- replating to new dishes (3 x 10^5 cells per plate)
- after the first passage, 80% confluency is quickly achieved (every 2 days)
Yes as you said your cells are too big. I think may be due to osmotic difference. How frequently are you changing the culture medium ?
Dear Mamatha, thank you for answering. I changed the medium 24 hours after plating the SVF in order to remove non-adherent cells (day 1). After that, I changed the medium every 2 days (day 3, day 5,...).
Hi,
Isolation of ADSC from rodents is accompanied with a period of dormancy. It means that your first passage of cells is not a pure population of cells. It is what I have experienced with ADSC isolation from black C57 mice. After 11 to 2d passage cells started to die and were removed by medium replacement. after 2-3 weeks some remaining cells started to proliferate. Flowcytometry and differentiation for these cells proved their multipotency and surface markers.
Overall morphology of ADSC is more flat compared to BMSC.
Good luck
Dear Maxim,
First of all, thanks for such a detailed question and clearing possible confusing answers which a layman can encounter while studying the question. I must appreciate that you have taken care of every minute detail while addressing your problem.
Coming to your question, as per my view you are worrying too much so early. Since your cells are dividing fine and you are changing media every third day they should be OK. Sometimes, cells do not reach their real size at passage 0 and it happens often in case of stem cells derived from animals. First they will be some small adherent bubbles and acheiving morphology slowly but not complete morphology is seen in passage 0. Once you trypsinize and replate at appropriate cell density then cells develop complete morphology. What I have observed that animal cells are comparatively smaller than human cells while culturing. On an average a BMSC derived from rat is 14micron in size while the same is 18micron in human. So comparing zero passage with 2nd or third passage will always give you misconception that your cells have become somewhat abnormally enlarged. As pointed out by Vahid, your population is not a pure one even at first passage and cells will become pure stem cells as you passage them. You can expect a pure population at 3rd or 4th passage and then you can caracterize your cells (as it is always recommended to characterize after/at P3). So if your cells are dividing fine, don't compare them with passage 0 cells. Let a pure culture be established and then measure size and that will be your prper size of cells. Sometimes cells become enlarged more than that size also but that happens only in case of stress conditions which is not the scenerio here.
Hope it helps !
Thank you for valuable answers, everyone.
Vahid Razban, what do you exactly mean by the "11" in "After 11 to 2d passage cells started to die and were removed by medium replacement"? Do you mean "After 11 hours"?
Ajay Kumar, indeed, characterising the cells will provide me with a lot more information than merely looking at the cell shape. I am planning this test soon. Can I ask you why you think the cells are not affected by stress in this case?
Thank you
Dear Maxim,
I think that because stressed cells don't divide much.They will just stay in culture like lousy heads and eat up media but will not divide ..As you have said that ur dish acheive confuence in 2-3 days, so I can say tht ur cells are not under stress for sure.
Cheers !
I repeated my experiment with younger mice (only 5.5 weeks old instead of 4 months) and with new stromal medium. They expanded very well with doubling every 2-3 days. I saw the same phenomenon with the cells increasing in size compared to when I plated the SVF, although it was less distinct. I did flow cytometry analysis after the third passage and it was positive for MSC markers CD73, CD90.2 and CD105. Other markers I tested were CD31, CD45, CD11b and CD34. These were are all negative (90% after staining with trypan blue. Thank you for the remarks you made.
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