Arifah, can you please clarify whether you are trying to isolate MSCs from the Wharton's Jelly at this point, or if you already have an isolated MSC population that you are trying to propagate? Are the cells you are using fresh from isolation, or revived from cryo? Also, what type of media are you currently using?

Media compositions for proliferation and differentiation are quite different. In addition, you will need different supplements for each differentiation pathway.

Dear Arifah

Previously I worked with WJ-MSCs and I used to isolate WJ-MSCs from umbilical cord tissue. 

To isolate from UC tissue, first I will clean the UC properly using normal saline water then I will cut UC in small around 2 inch pieces and will store in 50ml sterile falcon containing normal sailine added with penstrep to avoid/kill any contaminating agent at 4 degree. Later around after 1-2 hrs. we will open the tissue flaps around the arteries, remove the arteries which will expose the WJ tissue, cut that region and plate that in 35mm dish with DMEM low glucose, pen strep, FBS and glutamine media. Around 7th day you will be able to see the WJ-MSCs coming out through the tissue pieces. 

If you are simply taking out the frozen WJ-MSC vials from liquid nitrogen you are supposed to take extra precaution like do not immediately bring cells at room temperature as soon you take out cells from liquid nitrogen keep the vial at -80 degrees so the temperature goes up slowly, do it for around 10-15 mins, meanwhile make ready a beaker with sterile 37 degree Celsius distilled water. After 10-15 mins take out vial containing cells from -80, hold vial through a forceps while same time vial should be dipped in water at 37 degrees. Dip in water for 3-4 mins. Now the cells will be at normal room temperature you can add media and go for plating. These all steps are supposed to be performed in order to keep cells happy and to avoid damage caused by heat and shock. 

We never used to add ascorbic acid.

Make sure cells are not already dead when you are taking out vial from liquid nitrogen due to stress or other region.

I have experienced that WJ-MSCs can be passage up to 20 passages but proliferation will decrease as you go for more number of passages.

Be sure the samples you are working with are not contaminated.

I hope my suggestion will be helpful.

Hi Arifah,

We have been isolating and culturing WJ-MSC. More info you can find in Stem Cells Dev. 2015 Nov 15;24(22):2649-59.

Please note that the lot nr (batch) of FBS may be of great influence on your MSC culture. If you repeatedly cannot retrieve your MSC, try another batch or manufacturer.

The protocols for MSC expansion and MSC differentiation are remarkably different. For MSC expansion you just need DMEM, L-glut, 10-15% FBS and a bit of patience, because MSC growth may take a few days.

You don't need ascorbic acid to grow MSC. This (among other factors) you will need when you differentiate your MSC into the osteogenic lineage.

Good luck!

Best regards,

Meissa van Pel

Dear Arifah,

We isolated MSCs from adipose tissue and bone marrow mononuclear cells, I think that the former answers had a lot of information that probably will be useful. 

I am using revived cell from cryo which already in passage 3. Maybe i need to re-growth the cell in the small flask first instead of T-75 that i have been used before. This is the media composition that i have used for my stem cell growth into 500 DMEM-Low glucose Media :

1% of ascorbic acid

2% of HEPES

10% Fetal Bovine Serum

1% antibiotic-antimyotic

Just wondering is Glutamax function in this process?

. Thank you to all answer given. Highly appreciated and it really helped me in finding the solution.

Glutamax is a more stable form of L-glutamin. In a lot of culture media, L-glutamin is not added or just present at low levels. I fully agree with Santanu that Ascorbic acid is not very helpful in culturing MSC.

If your MSC don't grow too fast, or are thawed, it is better to seed them at high densities (5,000-6,000 MSC per sq cm). Once they start to proliferate, you can decrease the seeding (500-600 MSC per sq cm is the minimum).

Dear,

You can use the alpha MEM as culture media,some time, it takes more than 3 days to see the adherent cells.

Best of luck

Hi I agree with the suggestions above - seeding at higher cell density, using MSC pre-screened FBS, ascorbic acid not required, methodology suggested above for thawing. Just one suggestion .... after adding 1 ml media to the vial (and adding this 1 ml cell suspension to a T25 flask), take out a small aliquot (50 ul) and assess viable cells by trypan blue counting to see how many viable cells are present in your mix.

Best of luck!

Hi,

Higher cell density is proposed. Glutamax (preferred) or L-glutamine is mandatory.

How you revive the cells? Cells should rapidly reach 37 degree of centigrade. DMSO should be removed by centrifugation.

Good luck