Hello everybody,
Right now, my team and I are working on a research project that established a primary culture of mesenchymal stem cells from murine adipose tissue. We executed the establishment in April and, until this day, the cells have experienced low growth rates and difficulties forming colonies and monolayers.
We are using DMEM + HAMF12 as culture medium in a 1:1 ratio, supplemented with 10% of fetal bovine serum and 1% L-glutamine.
We have replaced the culture medium with a fresh one and carried passages when it has been necessary thinking that we would solve this issue, but we have not been successful yet. And, since they have not grown appropriately, we are not able to continue to the next step of the project.
I would appreciate your help and advice. Thank you so much!
Hi Josué,
When culturing MSCs, additional growth factors such as FGF2 or FGF4 with heparin are usually essential for MSC proliferation. For example, we cultured human placenta-derived MSCs with alpha-MEM/FBS/PS + FGF4, Heparin. FGF is a signal cue for MSC proliferation, while heparin helps FGF to work. Similarly, most studies treat FGF2 and other additional factors for MSC expansion.
MSCs in the body are in a quiescent state which refers to a low level of metabolism and proliferation until before some stimulation signals, which means that they have 'innate properties' with minimum proliferation and metabolism. This is why the additional factors above are required.
So, I guess checking culture methods from other papers using murine MSC, in perspective of additional supplement, would help you to solve the problem :)
Have a good day!
Yu
Years ago, before MSC were identified, I used to culture murine bone marrow stromal cells. In that culture system, the medium was RPMI 1640 + 5% FBS - NOT heat inactivated. Since then, I have worked with human MSC from bone marrow using the spicule fraction, and for that work, I used DMEM - low glucose/ 1 gm per liter + 10% FBS (also not heat inactivated).
Some key points I have found are 1) seeding density is important - at least for bone marrow stromal cells. They produce an autocrine factor [which turns out to be M-CSF] and at too low a cell density, they do not proliferate.
2) Passage before the culture reaches confluence is crucial - particularly for MSC. MSC undergo contact inhibition - a major decrease in gene expression - and once they are in that state, they grow painfully slowly. This is likely where bFGF comes in - as noted by Wondong Yu.
3) Use FBS that has not been heat-inactivated. The grade of the FBS seems to be important - not all products work for MSC cultures. You need to test suppliers. We have had good results with FBS from Sigma - catalog # F2442.
4) Using low glucose medium is important - at high glucose, you get an outgrowth of adipocytes. DMEM comes at either 1 gm per liter or 4.5 gm per liter. Use the 1 gm/liter DMEM.
One other point to note - DMEM/Ham's F12 at 1:1 is essentially the same as Iscove's Modified Dulbecco's Medium {IMDM].
Cite
1 Recommendation
Subhash C. Juneja
Johns Hopkins University School of Medicine
Addition of FGF2 is best. In my paper I compared FGF2 vs FGF2+WNT3A,
Only FGF2 was better for quality and growth of MSCs, does not matter it is human FGF2, it will work in Mouse also
Good luck, take my word
Cite
Josué R. Barquero-Chavarría
Institut Pasteur
Hi,
Thank you all for your advice, I really appreciate it. You all have given me a broader perspective in MSC cultures, I am going to apply your recommendations in upcoming assays and I will keep you updated on the matter.
Once again, thank you so much! Have a great day!
Cite
Similar questions and discussions
How can I avoid the self-activation of primary lung fibroblast during culture?
Question
5 answers
- Yinzhen Han
In the process of culturing primary fibroblast, my control group showed high level of a-SMA in subsequent Western blot, however, I need to confirm the activation effect of TGF-b, and the anti-activation effect of a drug.
What are these contamination in my Adipose tissue Mesenchymal stem cell culture?
Question
6 answers
- Satyajit Ghosh
I am culturing rat adipose tissue mesenchymal stem cell in DMEM low glucose 10% FBS media, after 1 or 2 days of culture I am getting this type of debris or contamination(shown in the picture: red circle). Please guide me are these contamination and why are these coming ?
What is the culture conditions of scc-9, fadu and detroit cell lines ?
Question
3 answers
- Elif Yaşar
I have tried different medium complements for each cell lines but there is a contamination that i don't know. So i couldn't expand them in culture. Which supplement i should use for each cell lines in medium.
How can I achieve large-scale expansion of human mesenchymal stem cells?
Question
2 answers
- Marcela Cárdenas-Tueme
Hello!
Im working with primary human MSC, I obtained them from adipose tissue, however, I've been trying to expand them unsuccessfully, I use normal T-75 tissue culture flask, DMEM with 15% SFB, is there any other strategy I could use? Do you have any tips you could share with me?
thank you
How to cultivate human fat tissue Mesenchymal Stromal Cells?
Question
Be the first to answer
- Oksana Novikova
Good afternoon, colleagues! I have a problem with cultivation of a culture of human adipose tissue Mesenchymal Stromal Cells. I have experience with animal fat MSCs cultures (rats, rabbits, mice, dogs, cats and horses), these cells divide and grow in culture very successfully. However, I worked with fat of 4 different donors of different age, but I cannot get much cells in vitro. To isolate cells, I use the enzymatic method, cultivate it in a standard DMEM/F12 medium with fetal serum. The cells attach to the plastic, spread out and undergo several divisions. However, after that, they begin to increase in size and give many shoots, after which they no longer divide. The addition of FGF does not change the situation. What could be the reason, what are your protocols for working with MSCs from human adipose tissue?
Can I run a qPCR experiment with gradient temperature or should my targets be amplified at single temperature ?
Question
6 answers
- Tuba Sena Ogurlu
Hello everyone,
I am running a qPCR assay. I chose gradient temperature option for each of my primer to get the best conditions the amplification happens (without heterodimers- NA in negative controls). However, I have seen that my housekeeping gene and one of my target gene have different annealing temperature. Can I run another qPCR set-up just for this gene by choosing gradient temperature option ? For instance; my gene in question in a row with 54C and housekeeping gene in a row with 60C. I think as far as the machine reads the signals at the same time, it won't pose a problem but I just want to make sure.
Many thanks,
Tuba
Is this appropriate media for growth of UM-UC-3 cells?
Question
3 answers
- Deleted profile
I have ordered UM-UC-3 cells and am wondering if the media I currently have is appropriate to use?
From the suppliers website, it states to use: EMEM EBSS + 2mM Glutamine + 0.1mM Non Essential Amino Acids (NEAA) + 10% Foetal Bovine Serum (FBS) + 1.5g/L sodium bicarbonate + 1.0mM sodium pyruvate.
We currently have MEM α, GlutaMAX with no nucleosides (https://www.thermofisher.com/order/catalog/product/32561037). This media has Glutamine, NEAA, sodium bicarbonate and sodium pyruvate already in it.
However, it was brought to my attention that this media mightn't be suitable to grow UM-UC-3 cells in and that I should instead use Minimum Essential Medium Eagle (https://www.sigmaaldrich.com/IE/en/product/sigma/m0325).
I was under the assumption that MEM α was just a modified EMEM that contains all the necessary extra supplements in it.
Is there any significant differences between the two?
How to run multiple samples in single run inq PCR with different anealing tempretures?
Question
8 answers
- Junaid Kori
I want to do qPCR of multiple genes but the problem is that the anealing temperature of multiple primer sets varies a little. How can i address this?
Related Publications
A three dimensional matrix for guided mesenchymal stem cell and urothelial cell growth and differentiation
Article
- C. Adelöw
- T. Segura
- J. A. Hubbell
- Peter Frey
The behavior of Wharton’s jelly mesenchymal stem cells in monolayer 2D culture condition
Conference Paper
- Maryam Haghighi
Differentiation of mesenchymal stem cells derived from bone marrow and adipose tissue into neural cells
Presentation
- Charbel Khalil
Got a technical question?
Get high-quality answers from experts.
Top contributors to discussions in this field
Mohammad Hassan Shahavi
Alejandro Martin
Pawel Buczkowicz
Arvind Singh
Robert Kiss
Join ResearchGate to find the people and research you need to help your work
- 25+ million members
- 160+ million publication pages
- 2.3+ billion citations
or
- Join for free
- LoginEmail Tip: Most researchers use their institutional email address as their ResearchGate login
PasswordForgot password?
Keep me logged inor
Continue with Google
Welcome back! Please log in.
Email · HintTip: Most researchers use their institutional email address as their ResearchGate login
PasswordForgot password?
Keep me logged in
or
Continue with Google
No account? Sign up
- Badges
- Science topic