Material and Methods 12 2.1.4. Adipogenic differentiation For adipogenic differentiation, 5,000 cells/cm 2 in passage 2 were seeded in a 6-well cell culture dishes and expanded to 100% confluence. Adipogenic differentiation was induced for 5 days with D-MEM high glucose media, 10% FBS, 40 IU/ml penicillin/streptomycin, 1 µM dexamthason, 0.2 mM indomethacin, 0.1 mg/ml insulin and 1 mM isobutylmethylxanthine (IBMX), all from Sigma-Aldrich, followed by conservation media (D-MEM high glucose media, 10% FBS, 40 IU/ml penicillin/streptomycin and 0.1 mg/ml insulin) for additionally 2 days. Induction / conservation cycle was repeated for 3 weeks. Cells in standard culture media (α-MEM) served as a control. Experiments were performed twice in triplicates. After 21 days of differentiation, cells were washed with PBS (with Ca 2+ & Mg 2+ ), fixed with 4% PFA for 10 min at RT and thereupon stained with 0.2% Oil Red O solution for 20 min at RT. After washing three times with distilled water, cell monolayer was imaged with histological phase contrast microscopy as described in 2.1.3. For quantification of lipid accumulation in fat vacuoles, cells in an additional 6-well were washed with PBS (with Ca 2+ & Mg 2+ ) and 5 ml PSB/140 µl AdipoRed TM solution (Lonza, Walkersville, USA) was added to each well according to the manufactures protocol. After 15 minutes of incubation fluorescence values was quantified using a UV/VIS multiplate reader (Safire 2 , Tecan, Männedorf, Switzerland) at a emission of 572 nm. 2.1.5. Surface maker analysis According to the consensus of the International Society for Cellular Therapy by Dominici et. al. mesenchymal stem cells are required to express CD105, CD73 and CD90 (≥ 95%) and cells must be negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR (≤ 2%) 19 . Each antigen used to verify the expression of surface markers is presented in detail in table 1. HMSCs in passage 6 were trypsinized, washed in warm PBS and subsequently 10.000 cells per cluster of differentiation (CD) marker were resuspended in 200 µl PBS and transferred to FACS tubes (BD Falcon TM , Heidelberg, Germany). Finally 3 µl of primary labelled antibodies or isotype controls (for detailed descriptions see Tab. 9: Used chemicals & cell culture media. on page 59) were added and incubated 20 minutes at RT in darkness. Subsequently, cells were centrifuged at 500g for 10 minutes and resuspended in 200 µl PBS. Fluorescence signal was measured by a fluorescence activated cell sorting (FACS) de-
Material and Methods 13 vice (FACSCalibur, BD Bioscience, Heidelberg, Germany). Acquired data was analyzed using FlowJo FACS analysis software version 7.6 (Treestar Inc., Ashland, USA). Cell fragments were excluded by gating prior experimental evaluation and CD markers were set to approximately 95% fluorescence intensity of each isotype controls. Tab. 1: Detailed description of CD markers. CD marker full name, description CD105 endoglin (aka MAb SH2) CD73 ecto 5’ nucleotidase (aka Mab SH3 & SH4) CD90 cell surface protein Thy-1 CD45 protein tyrosine phosphatase CD14 membrane glycoprotein CD19 B-lymphocyte surface antigen B4 HLA-DR human leukocyte antigen receptor 2.2. Cell characteristics 2.2.1. Cumulative population doubling To examine the possible influence of the different culture conditions on cell proliferation, the cumulative population doublings (cum PD) and therewith population doubling time (PDT) were determined until cells went into senescence. From passage 1 upwards, 50.000 cells were seeded in a T-25 cell culture flask. After 4 days cells were trypsinized and the cell number was assigned using a cell counter (Cedex XS, Roche Innovatis, Bielefeld, Germany). Consecutively, 50.000 cells were transferred into a new cell culture flask until cum PD reached a plateau phase. To ensure that cells develop a prolonged population doubling time, trypsinisation interval was enhanced to 6 days. Measurement of the cum PD was stopped when it reached a decreasing cell number. Cum PD was calculated as following: cum PD=ln [NE/NB] / ln 2 (NE: end cell count; NB: cell count in the beginning).
List of figures 63 List of figures
List of figures 65 Fig. 18: Compari
Acknowledgments 67 Acknowledgments