MTT assay MC3T3-E1 Osteoblast metabolic activity in dependence on different supplements Kathrin Smuda, Radostina Georgieva and Hans Bäumler Charité-Universitätsmedizin, Berlin Institute of Transfusion Medicine and Berlin-Brandenburg Center for Regenerative Therapies ( BCRT)

2 Overview Introduction –(Pre-)Osteoblasts –What is a MTT assay? –Question / experimental design Results Conclusion / Outlook

3 (Pre-)Osteoblasts MC3T3-E1 –Pre-osteoblasts –Mus musculus –Bone/calvaria –Adherent growth –Fibroblast morphology –model osteoblast cell line 25 µm Introduction

4 MTT assay MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Colorimetric assay Determines the cellular metabolic activity / cell vitality MTT is reduced to its purple and insoluable formazan in living cells (metabilic active cells) Reduction of MTT increases with cellular metabolic activity Dissolving of formazan results in a coloured solution; absorbance can be detected Introduction

5 Question Osteoblast growth/vitality/metabolic activity depending on different supplements. How do PRP (+ Leucocytes), PRP and PPP affect osteoblast vitality in a MTT assay? PRP – platelet rich plasma PPP – platelet pure plasma Introduction

6 Experimental design ALP testMTT test Differentiation: Osteoblasts Seeding Pre-Osteoblasts + Ascorbic Acid Introduction

7 Experimental design PRP (+ Leucos)PRPPPP platelet concentrate whole blood (citrate as anticoagulent) sedimentation for 2 h x g 15 min w/o collagen 1 µg/mL collagen w/o collagen 1 µg/mL collagen w/o collagen 1 µg/mL collagen Introduction

8 Intermediate Results Differentiation (ALP assay) pre-osteoblastsosteoblasts ALP negativeALP positive Results

9 Cytoskeleton stained with Alexa Fluor® 488 Phalloidin; Nucleus stained with TO-PRO®-3 Iodide Confocal microscopy of osteoblasts

10 Results

11 Results Significant difference between control and PRP (+ Leucos) w and w/o collagen Significant difference between w and w/o collagen

12 Results Significant difference between control and PRP (+ Leucos) w and w/o collagen Significant difference between w and w/o collagen

13 Results No significant difference between control and PRP (+ Leucos) w and w/o collagen Significant difference between w and w/o collagen

14 Results No significant difference between control and PRP (+ Leucos) w and w/o collagen No significant difference between w and w/o collagen

15 Conclusion Plasma itself cannot replace cell culture media Platelets release growth stimulating factors: Platelet derived growth factor (PGDF) Leucocytes seem to have additional growth stimulating impact Decreased reduction of MTT after 96h –Metabolic products / dying (sedimented) cells built sticky layer –decreased gas exchange –Detachment of the cells Conclusion

16 Outlook Upcoming experiments –Investigate if platelet rich plasma containing leucocytes can fully replace osteoblast cell culture media in the long-term –Distinguish between activated and inactive platelets and determine the effect on osteoblast growth –Avoiding sticky layer by using inserts like ThinCert™ cell culture inserts ( Outlook

17 Acknowledgements Research group of Hans Bäumler –Radostina Georgieva –Michael Koziol –Yu Xiong Max-Planck Institute –Christine Pilz PIRSES-GA KF AK0 KF FR0 KF AJ2