Hello Fawad. The rat Bone Marrow MSC shouldn't behave like that. My rat BMSCs under proliferation conditions use to reach confluence in 48h maximum (I culture them in T75 flask). So, I wonder if your DMEM/F12 is already supplemented with some Glutamne source (Glutamax or L-Glutamine), if is not, probably is the lack of L-Glutamine and you should add 1% L-Glu to your medium. Another key point is the density of cells, MSCs behave different depending of the density, in my opinion the best density to growth MSCs is about 6700 cell/cm2, lower than that use to cause slower rate of replication and also phenotype's changes. But, if you increase so much the density they start the selfdiferentiation process. Another possible reason is that your cells suffer some damage dureng the cryopreservation or the thawing process, the MSCs are very sensitive to these two processes.

I hope you will find the solution to your problems with the MSC. Good luck.

Arlyng 

Dear Fawad,

The BMMSC from rats need some growing factors like: IGF-I or TGF-ß as well you need to start with 5x100000/mL. We used the same DEmen/F12 with low or high glucose as well FCS in 10 percent!

But those cells growing less than same human fraction!

Katherine.

Dear Fawad!

Maybe this thread can help you, there are a lot of useful tips and answers to a similar problem.

greets, Angelika

https://www.researchgate.net/post/My_MSC_cultures_started_floating_and_detaching_after_2_days_of_culturing_Can_anyone_help_me11?_sg=2Mr8iHL831%2FMGdZLlLfKRDXZxfYonv7oQWhhyu9ebgtkU2c2xDsHKO8Z42jKQ4Pp_iP0PRK4%2FciWM9pwfg6np1XnyqZemMBeQFB13VXfmMq6DkDQjAV%2FBgmMf0fu30Adw_wnkr7J9f7hfs8TaNJR212TBRKc0jSw6P2ZyP1X9fVzBfE8JgOT%2B7YL%2B5s%2FT0HD0p#view=4fb23fe3e4f0764c6c000000

Dear Fawad,

I have more experience with human MSCs, but one of the critical aspects about culturing MSCs is the quality of the serum. We always use MSC-qualified serum in our cultures. Althought it is considerably more expensive, it works just fine, at least for every batch we've used so far. These lots are tested by the manufacturer, but you can test several batches of regular serum yourself, but I have the impression that it might end up being even more expensive and for sure time-consuming.

You may also want to test alpha-MEM (no nucleosides) instead of DMEM/F12. The glutamine issue is also important (2mM glutamine should do it).

Best regards,

Mário

Hi Fawad, 

I agree with Mario, you should try using alpha-MEM. In my experience, culturing rat bone marrow MSC in DMEM/F12 did not yield good results. With alpha-MEM we were able to grow them up to the 9th-10th subculture without changes in cell morphology or proliferation rates.

Good luck 

do you detach the cell using scrape?

it is a very critical factor for successful culturing of MSCs. so i will suggest to work on optimization of sub-culturing protocols along with using alpha MEM with 10% FBS

Use Alfa MEM with 15% MSC qualified FBS. You will see much better growth after fifth passage when rest other cells die only MSCs proliferate. 

Dear Researcher

Let me know did you direct approach or indirect approach?

in my experience FBS matters a lot as someone else stated.  We use 15% FBS from Sigma (which is MSC qualified I believe).  Using standard 10% FBS dramatically slows growth.  

Hello

Preferably using 45% alpha-MEM and take into account initial plating densities , we use this protocol for isolation MSC.

1- Briefly marrow flushing from femurs and tibias with Hank’s buffered saline solution

2-centrifuge at 600 × g for 10 min.

3- Cells is resuspending in 45% Ham's’ F-12

4-45% α-MEM

5-10% FBS

6-supplementing with 100 U/mL penicillin G and 100 µg/mL streptomycin sulfate

7- plating into T75 flasks

8-removing and adherent cells non-adherent cells is detaching with 0.05% trypsin/0.53 mM EDTA , After 8 days

9- After 3 days, cells is detaching with 0.1% trypsin/0.02% EDTA (passage 1). MSC is storing in 30% Hams’ F-12, 30% α-MEM, 30% serum, and 10% DMSO in liquid nitrogen.

In attachment two papers , I hope to be helpful

Regards

thanks a lot to all of you for valuable comments. I shall just start optimization for each protocol and try to get best results.. 

thanks to all for valuable time and guidance

best regards    

Hello Fawad, my bone marrow cells are quite fast at growing. First check how old the rats are, the older they get the cells tend to slow down a little. Once you have your cells seeded, first of all one or 2 feeds with low glucose dmem so that you select out the non msc's then once you achieve that start feeding the cells with the usual dmem you have been using and they should catch up with growth speed

thanks Antonio M Spadaccino for your kind reply... actually I am using rats with induced disease in young Age,,, and I am not having any low glucose DMEM medium, u can suggest me some if possible..

thanks 

Hi Fawars,

I was in a similar situation. After some years growing MSC from rats the cultures started to going down after the first/second passage. This happened after buy a new FBS batches. The pretested FBS works well but is expensive... So, to solve my problems I asked some companies for a sample of FBS from different batches. I tested the FBS samples from different companies and different batches in the same culture and I quantified the intra-passage growth speed and the accumulative between passages. Finally, I choose the best one and reserved enough bottles of this batch with the correspondent company to ensure the FBS for long time. Then, the FBS batch is very important not only for the MSC but also for other types of cells since every batch can be different to other.

Other important thing as Antonio said is the animal age since the growth ratio of MSC from young animals is higher than old ones. Moreover, in your animals with this induced diseases it is possible that this diseases affects the MSC characteristics similar to other diseases. http://www.ncbi.nlm.nih.gov/pubmed/22194749

I normally use low glucose DMEM with glutamax from invitrogen, the basic one is good enough. As others suggested add some glutamine or I tend to use more serum, so go from 10 to 15% maybe that will speed up cells. I am not sure if the slow growth is actually linked with the disease somehow. I normally purify, seed and 3 weeks later find stem cell. Once they can be visualised with the microscope (30/40% density) they quickly catch up and need to be split 1 in 3 after about a week. 

thanks  Abel Torres Espín and  Antonio M Spadaccino for kind suggestions.. 

 Hello Fawad  try to use DMEM medium with low glucose content with 15% MSCs qualified FBS ,avoid using   rats with induced disease for isolation ,