Are they primary MSC or immortalized? If primary, it is known fact that they can not be kept in culture for more than 3-4 passages, because they transform and/or undergo senescence. I usually split them ones a week and keep them in culture for 1 month, then take a new vial. Another thing: never let them go confluent like in your picture, they lose pluripotency.

Your cells are senescent. I suggest you to calculate "cumulative number of population doublings" (cPDs) for each passage and draw the growth curve cPDs vs culture time. In this way you can have informations on your culture phase (exponential growth, static growth or senescence) passage by passage. To calculate cPDs at a specific Passage:

cPDs = 3.32 (log X - log Y) + Z

where, X: number of cell harvested ; Y: number of cell seeded and Z: cPDs at previous passage. Remember to perform any experiment with your MSC in exponential growth phase.

In my experience, you can have several problems with your culture conditions which may be affecting your cells.

Cell donor as well as passage, if your MSCs are from a primary culture, are key factors in survival and proliferation rates. Donor Age is strongly related to proliferation rates, being diminished as donor age increases. Also, low passage is critical to perform reliable experiments in which cells maintain pluripotency.

You need to be careful with the exposure time to trypsin, and try it to be the shortest possible because it can negatively affect your cells. Freezing conditions are also critical for cell survival when thawed. 

In order to confirm senescence of your MSCs in your culture conditions, you can perform a Beta-Galactosidase Staining which tells you if senescence is actually happening in you flask or it is another kind of cell death. When looking at the pictures, I would say you have some senescent cells, but definitely not all of them. It is common to have heterogeneity in your flasks because some cells are older than others but I would perform the assay to discard the cells if senescence is ocurring.

Everything I have just written is only according to my experience with BMSCs and ADSCs, and may not help you to resolve your problem, but I hope it will be useful to consider your options.

Regards

Eva

Can anybody suggest me why I get a thick white clump after trypsinization followed by centrifugation of Mesenchymal stem cells? how can I get rid of it? because there are no cells when I count.

Sometimes I get the same tick withe clump that you are mentioning MOHD AQDAS, and no cells or very few when you count with out this clump. I do not know exactly why this happens but I really think this happens when cells are confluent and touching. They kind of secrete extracellular matrix or something and form this non disocciable clump. 

Eva Rubio, thank you very much for your help. Do you have a protcol of the Beta Galactosidase staining that you could share with me?

http://www.cellsignal.com/product/productDetail.jsp?productId=9860

This is the kit I use. It works quite well. You just have to follow instructions. Its easy to perform.

Regards

@jessica: thank you dear. what exactly have you done in order to get rid of the thick clump???

@mohd I try to trypsinate them way before confluency like 70%... whenever I trypsinate them before confluency I do not have the clump problem... BUT I have the problem of very low yields.. they almost do not grow.. 

its almost the same am too facing dear... yield is a huge problem 

I have the same problem with primary BM stromal MSC culture from STEMCell Inc. They grow painfully slow and each T75 yeilds only 1million (sometimes less) cells.

And at P4-5 my cells already grow slow. Now I try to keep my culture under P4. And the lot/donor variability can be huge. I have 3 lots, one grow exceedingly well until P6, the other significantly slowed down at P3-4. I am working on my 3rd lot, which doesn't look exciting so far.