thaw your serum and keep it in 56°C water bath for 30 minutes, swirl to have homogenized heat distribution.  Cool it down on ice and aliquot and transfer to a -20°C or use it directyly.

You can try MSC qualified Serum as it supports canine MSCs

https://tools.lifetechnologies.com/content/sfs/brochures/F066604_MSC_FBS_F.pdf

I don't know about canine, but for for human MSC you can use Nutristem media with its supplements (without serum). Otherwise if you look for a cheaper solution: DMEM-F12, 10% FBS (heat inactivated), supplemented with Glutamine. In both case MSC stay undifferentiated, but with DMEM-F12 the cells growth is very slow and the cell morphology in the two media look different.

No experience with canine, but with different subsets of human MSCs we always used native (non heat-inactivated) FBS Gold from PAA, 10% in DMEM-LG. You cannot have spontaneous differentiation with this kind of media, nor losing any differentiation potential.

We chose (till it was available here) this kind of serum as being quasi-defined in its composition. Now there are problems for sourcing it in EU 8due to marketing reasons of the controller of PAA), but maybe it may be sourced more easily in US.

Never had problems with non heat-inactivated serum.

I use heat inactivated serum (at 56°C for 30min), which works fine, and never tried KO serum so far. Good luck.

The spontaneous differentiation seen in cultures of stem cells grown in serum has nothing to do with the stem cells themselves per se. Rather, what is occurring is that most serums induce differentiation of uncommitted stem cells in culture because of inductive factors present in the sera. Unfortunately no two lots of serum have the same composition and/or concentrations of inductive factors. The easiest method to chose a supplement for your culture media to maintain the cells in an undifferentiated state is to run reciprocal supplement dilution curves in 96-well plates. To accelerate phenotypic expression, add 2 ug/ml insulin (a progression agent that accelerates the phenotypic expression of differentiated cells but has no inductive activity). Additionally, if you want to differentiate the cells to see their "stemness" potential, save the supplement that failed the above analysis miserably and you will have an excellent agent to test your cells for their stemness by induced differentiation.

See attached article for a more detailed explanation.

Dear Litia Alves de Carvaiho, I would agree yet disagree with your statement about using serum with stem cells. On one hand if you want to determine the stemness of one particular stem cell within a mileau of precursor (stem and progenitor) cells, then by all means use single factors for testing. We characterized single cell-derived clonal populations of our stem cells and progenitor cells by that method. In the process we also learned that the same growth factor would show different activities based its concentration (effective dose) on the type of precursor cell analyzed, be they individual lineage committed progenitor cells, germ layer lineage stem cells, pluripotent stem cells or totipotent stem cell. For example, TGF-beta has been reported in the literature in numerous publications to stimulate proliferation in stem cells. We tested serial (micromolar to phenotomolar) concentrations of  recombinant TGF-beta with single cell-derived clones of stem cells, i.e., totipotent stem cells, pluripotent stem cells, mesodermal stem cells; and progenitor cells, i.e.,  myoblasts, chondroblasts, adipoblasts, osteoblasts, and fibroblasts. We used culture media containing either serum-free supplements or heat inactivated serum to negate the effects of any potential contaminating inductive, proliferative, progressive and/or inhibitory activities. in general the results demonstrated that while the progenitor cells would proliferate under the influence of TGF-beta, the stem cells would not, but actually committed to the fibrogenic lineage. The results also demonstrated that the TGF-beta displayed different activities based on the concentration that the precursor cells were incubated within.

A great many scientists spend their whole research careers looking at the activity of a single cell type or a single organelle or a single gene or a single bioactive factor, etc. out of context of the body as a whole. But you need to remember that serum is a by-product of circulating blood. And it is that liquid element of blood that is bathing resident precursor cells and differentiated cells on a continual basis within the body. If one is to recognize the many interchanges and interactions that occur within an individual with respect to their differentiated cells, progenitor cells, stem cells, extracellular matrices, autocrine factors, paracrine factors, endocrine factors, cytokines and bioactive factors, then one also needs to ascertain how they interrelate to one another when nourished by multiple proliferative, inductive, progressive, and inhibitory agents. In this instance it is cost-effective to use serum containing (pre-characterized) active bioactive factors than trying to recreate what is normally present at the appropriate concentrations in the individual. 

See attached article for a more detailed explanation.

Dear Litia, What I said was that the use of serum depends of the particular studies you are doing. If you want to test individual entities, then do not use serum, there are just too many unknowns. However, if you want to understand synergistic actions, first characterize the components in the serum as well as their concentrations. As long as you know all the "players" involved, i.e., cells, ECM, bioactive factors, etc.,then one can perform reciprocal studies and generate meaningful data.  

I understand what you are saying, but please hear me out. Just prior to our endogenous adult stem cell studies, which were initiated over 25 years ago, I was attempting to isolate a glycoprotein derived from bone matrix that would stimulate skeletal muscle formation in vivo. At that time we initially developed a biological assay utilizing myoblast progenitor cells and our ELICA procedure. This combinatorial assay would quantify the biological activity of muscle induction in vitro. We had developed an assay to measure defined units of biological activity. At that time we using a lot of serum that had been left out on our loading dock, in the sun, for over two weeks. We noted that that particular lot of serum demonstrated zero biological activity at any concentration examined. We utilized that serum in our culture medium to partially purify the aforementioned glycoprotein. The data is published in the Cell Biochem Biophys paper. We were nearing the end of that lot of serum and began testing other lots of serum for biological activities. Our test utilized a reciprocal 96-well plate format in which a known inductive factor is paired at reciprocal serial dilutions against a serum sample to be examined. At a concentration of 10% v/v serum in culture medium this particular lot of test serum inhibited the inductive activity of all recombinant factors in our inventory. Because of our unique assay for quantitative biological activities we were performing contract testing at that time for inductive recombinant growth factors from Sigma, Cal Biochem, R&D Laboratories, Genetics Institute, Amgen, P&G and a few other companies. We hypothesized that this particular lot of serum contained an inhibitory factor to inductive activities. LIF (leukemia inhibitory factor) was routinely used at that time to inhibit the spontaneous differentiation of ESCs. We tested this lot of serum head to head with LIF and discovered that it outperformed LIF for maintaining all types of uncommitted stem cells in an undifferentiated state. Thus we had discovered a compound within this lot of serum which we designated as ADF for anti-differentiation factor. We used ADF, first in the serum form and later in the recombinant form to prevent lineage commitment in our stem cells. As hopefully you can surmise, the two week sun exposed serum was essentially heat inactivated serum. Thus, we had developed two very powerful research tools that were based on the activities of serum. Therefore, use of serum can be a very powerful research tool if one just “thinks outside the box”.

If the individual wants to maintain stem cells in the undifferentiated state and the cost of knock-out serum or serum-free defined medium is just too pricy, they could use either LIF, which works at cell concentrations up to confluency (for progenitor cells and germ layer lineage stem cells) or ADF which works below or above confluency (for progenitor cells, germ layer linage stem cells, pluripotent stem cells or totipotent stem cells). Either one of these inhibitory factors can be used with serum to propagate stem cells in an undifferentiated state.

And thank you, Litia, for the verbal interaction. I hope we meet each other at a meeting some time. Take care.

Mesenchymal stem cells have been cultured since a long time, much before pluripotent stem cells. During those days people did not have an access to knock-out serum and had always used heat-inactivated serum. Therefore, if you are a new researcher and the studies do not involve any clinical applications, it is OK to use heat inactivated serum from bovine origin for culturing MSC.

Dear Litia,

I am working on dental stem cells and have worked with both heat inactivated and KSR (knockout serum replacement).

As you can also look into my publication that dental stem cells are quiet stable and don't get differentiated spontaneously so easily. So using a heat inactivated serum (the traditional way) will not result in spontaneous differentiation untill and unless you use a good quality FBS (preferably US origin). In case of very gud quality serum you don't even need to go for heat inactivation procedure but that can compromise antibody binding during some immunostaining procedures.  Dental stem cells work fine with a-MEM, so choice of media is also very essential in culturing cells. KSR is a good option but a costly affair too. KSR can be used when you go for certain differentiation protocols (neural differentiation -1-2%) and it works really fine there. So according to my experience, For culturing, proliferation and maintaining a naive differentiated state, Heat inactivated US origin FBS along with a-MEM will work fine in your case .

Hope it helps !

Using Knock out serum replacement with recombinant proteins would be ideal, but expensive. You can use heat inactivated FBS to expand MSCs, but keep regularly checking the protein and RNA profile to ensure that MSCs are not differentiating. On Knockout serum, it tends to more BMP, which can disrupt some cells types. Good luck with using heat inactivated FBS.

Alternatively, Atlas Biologicals in Fort Collins CO make a heat inactivated serum that works wonders. Previously, I was attempting to explain trouble-shooting. If you just want a serum that will do the job at a reasonable cost and forget about trouble-shooting your cultures then the heat-inactivated serum from Atlas is superb.

Heat inactivated serum worked well with human mesenchymal stem cells