Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use.

Mesenchymal stem cells (MSC) are promising candidates for cell-based therapies. One major obstacle for their clinical use is the biosafety of fetal calf serum (FCS), which is a crucial part of all media currently used for the culture of MSC. Nine donors each contributed 5 mL of bone marrow aspirate. Isolation of MSC was conducted according to Caplan et al., although for expansion we used low-density seeding with 20 MSC/cm2. Four different media A, B, C, and D were tested, containing 1%, 3%, or 10% autologous serum (AS), or 10% selected FCS, respectively. MSC were cultured on 24-well plates until passage 2 and counted under the microscope at regular intervals. Osteogenic and adipogenic differentiation were induced in vitro by using a modified standard cocktail and were evaluated semi-quantitatively through a microscope. Isolation of MSC after 3 days appeared best in media C with almost always C>D congruent with B>A. Proliferation was exponential with generally C>D>B>A. Morphologically, MSC isolated and expanded in medium C were indistinguishable from those in medium D. Phenotypic markers of MSC grown in medium C were: CD34-, CD45-, CD90+, CD105+, MHC class I+, MHC class II-, similar to MSC isolated and grown in medium D. Moreover, MSC grown in medium C showed more osteogenic potential than those from medium D in all cases: C+++, D++, B+, A 0. Cells retained their immaturity as shown by adipogenic differentiation and it always was: D+++, C++, B+, A 0. Growth of MSC in a FCS-free medium is feasible without addition of growth factors. Ten percent AS appears at least as good as 10% FCS with regard to both isolation and expansion of human MSC, while 1% and 3% AS appear inferior. With respect to osteogenic differentiation, 10% AS proved superior to the other serum conditions.