Transcript Presentation

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)
Overview
• Introduction
– (Pre-)Osteoblasts
– What is a MTT assay?
– Question / experimental design
• Results
• Conclusion / Outlook
2
Introduction
(Pre-)Osteoblasts
• MC3T3-E1
–
–
–
–
–
–
Pre-osteoblasts
Mus musculus
25 µm
Bone/calvaria
http://www.flickr.com/photos/cambridgeuniversity-engineering/4708106047/sizes/o/in/photostream/
Adherent growth
Fibroblast morphology
model osteoblast cell line
3
Introduction
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
http://www.biolab.cn/uploads/1/Image/20090606032015489.jpg
4
Introduction
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
5
Introduction
Experimental design
Seeding Pre-Osteoblasts
+ Ascorbic Acid
Differentiation: Osteoblasts
ALP test
MTT test
6
Introduction
Experimental design
whole blood
platelet concentrate
(citrate as anticoagulent)
sedimentation
for 2 h
PRP (+ Leucos)
w/o
collagen
1 µg/mL
collagen
4.000 x g
15 min
PRP
w/o
collagen
1 µg/mL
collagen
PPP
w/o
collagen
1 µg/mL
collagen
7
Results
Intermediate Results
• Differentiation (ALP assay)
pre-osteoblasts
osteoblasts
ALP negative
ALP positive
8
Results
Confocal microscopy of osteoblasts
•Cytoskeleton stained with Alexa Fluor® 488 Phalloidin;
•Nucleus stained with TO-PRO®-3 Iodide
9
Results
Osteoblast metabolic activity [%]
24h [%]
48h [%]
72h [%]
96h [%]
350
300
250
200
150
100
50
0
Co
ro
t
n
l
+L
P
PR
eu
/o
w
s
co
ll
Co
eu
L
P+
R
P
w
s
co
ll
ll
Co
P
PR
w
/o
Co
P
R
P
w
ll
Co
PP
P
o
w/
ll
Co
P
PP
w
ll
Co
10
Results
• Significant difference
between control and PRP
(+ Leucos) w and w/o
collagen
• Significant difference
between w and w/o
collagen
11
Results
• Significant difference
between control and PRP
(+ Leucos) w and w/o
collagen
• Significant difference
between w and w/o
collagen
12
Results
• No 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
• No significant difference
between w and w/o
collagen
14
Conclusion
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
15
Outlook
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
(http://www.greinerbioone.com/nl/belgium/articles/news/42/)
16
Acknowledgements
• Research group of Hans Bäumler
– Radostina Georgieva
– Michael Koziol
– Yu Xiong
PIRSES-GA-2013612673
• Max-Planck Institute
– Christine Pilz
KF2330502AK0
KF2354402FR0
KF3042301AJ2
17