Maximizing the osteogenic potential of stem cells after expansion in DMEM LG using 99mTc-HDP Labeling

Objectives Dulbecco's Modified Eagle Medium low glucose (DMEM LG) is one of the most important standard cell culture media used for tissue engineering of human mesenchymal stem cells (hMSC) worldwide. Until today it’s pretty far unknown which media is to favor after a DMEM LG based cell expansion to maximize the osteogenic differentiation. We developed a novel method to track and quantify the amount of newly produced hydroxylapatit using 99mTc-HDP, as the Diphosphonate binds to newly produced hydroxylapatite by human mesenchymal stem cells in vitro. As we could show in prior studies 99mTc-HDP Labeling is a new, high significant and accurate method.

Methods We have evaluated, after cell expansion in DMEM low glucose, the osteogenic potential of five basal cell culture media in combination with various growth factors. Thus we are able to determine the optimal combination of expansion-, and differentiation media to maximize the production of bone-specific mineral (hydroxylapatite) by these cells. The following media combinations were evaluated: 1) DMEM low glucose 2) DMEM high glucose; 3) Alpha-MEM; 4) DMEM-F12; 5) Verfaillie-Media. The cells were cultured in monolayer 35mm petri dished. After cell culture 5,5 MBq 99mTc-HDP was applied to the dishes, solved in 0,9% NaCl solution. The cultured were incubated for 2 hours followed by repeated washing. Then, the probes were placed under a gamma-camera, using a 256x256 grid. Gammacounts were acquired for 180 seconds. Using regions of interest, the counts emitted by each dish were calculated. A corresponding non-osteogenic negative control was cultured for each donor and group.

Results Cell expansion with DMEM low glucose followed by osteogenic induction with media (5) ,,Verfaillie“-Media showed the highest osteogenic potential over all in the evaluated media combinations with 47328 counts/180 seconds. The corresponding negative control hand an uptake of only 3316 counts/180 seconds. The second highest uptake was noticed for DMEM LG as induction media with 39356 counts/180 seconds, the corresponding negative control had an uptake of 3807 counts/180 seconds, followed by 30847 counts/180 for induction in DMEM HG (negative control 4981 counts/180 seconds) and Alpha-MEM with 28948 counts/180 seconds (negative control 2945 counts/180 seconds). The lowest uptake was shown for osteogenic induction in Berner Media with 2026 counts/180 seconds (negative control 3381 counts/180 seconds). Significance was set to p < 0,05 for students t-Test-analysis. In direct comparison, all osteogenic groups showed a significant higher uptake for the tracer in the osteogenic group compared to the negative control group, except for the Berner media where no significant difference could be calculated.

Conclusions All four media combinations worked very well to archive osteogenic differentiation, except the DMEM-F12+growth factors. Here no osteogenic differentiation could be archived. To get the maximum amount of hydroxylapatite, ,,Verfaillie“-media should be used. As the ,,Verfaillie“-media is a very expansive cell culture media, we will favor DMEM low glucose for the osteogenic induction after cell expansion in DMEM low glucose in the future. DMEM Low Glucose for osteogenic differentiation of human mesenchymal stem cells is still an economic and highly potent cell culture media to favor the deposition of hydroxylapatite.