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SSBBR presentation

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Pooja Jairam

on 25 July 2014

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Transcript of SSBBR presentation

Optimizing the Yield of Mesenchymal Stem Cell Purification from Bone Marrow
Optimizing the Yield of Mesenchymal Stem Cell Purification from Bone Marrow

Pooja Jairam
7/26/13
Dr. Dan Link and Dr. Bryan Anthony
Introduction
Mesenchymal stem cells (MSCs) are multipotent cells that have many functions such as supporting hematopoeitic stem cells.



Methods - Flushing, crushing, centrifugation experiment
Conclusion
Results and analysis continued
Methods and Results - enzyme incubation period for bone experiment
Methods and Results - enzyme incubation period for bone marrow experiment
Results and analysis
Acknowledgments
Link Lab: SSBBR:
Dr. Dan Link Program Director - Amy O'Brien
Dr. Bryan Anthony
Amy Schmidt
Ryan Day
Mahil Rao Howard Hughes Medical Institute
Grazia
Molly Washington University School of
Josh Borgerding Medicine
Jun Xia
AJ
Maria Trissal
Dr. Terrence Wong
Tim Supakorndej Profusing techniques: Dr. Jessica Anthony
Iris
Ricardo
Dr. Laura Schuettpelz
Josh Borgerding
Molly

Flow cytometry
Obtained from http://www.bu.edu/flow-cytometry/
Results show that centrifugation results in greatest yield of MSCs, but difference is not statistically significant. Difference between crushing and flushing is statistically significant.
The differences between the methods and time points are minimal.

Because this data can be considered biologically significant, we can conclude that centrifuging the cells, treating the bones with collagenase for 1 hour, and treating the marrow with collagenase for 10 minutes results in the highest yield of MSCs.
centrifuged
crushed
flushed
MSC
osteoblast
chondrocyte
adipocyte
Purpose
MSCs are extremely rare (1 in 50,000)

Goal: Purify MSCs in order to perform further experiments on them, such as analyzing genes using a gene chip.
Optimize MSC purification
-Method of bone marrow extraction
-Flushing, crushing, centrifugation
-Collagenase digestion optimization

MSC identification
Sca1
PDGFRa
p < 0.05
Marrow

Bones +
collagenase
Incubation periods:
10 minutes
30 minutes
60 minutes
The data is not statistically significant, but there is a trend that seems to show that the hour incubation period results in the greatest yield of MSCs.
Bones
Marrow +
collagenase
Incubation periods:
0 minutes
10 minutes
30 minutes
60 minutes
Extraction of Bone Marrow from mice
-Mice were sacrificed
-Femur, tibia, and upper pelvis of both legs were removed
-Either flushing, crushing, or centrifuging



Future Directions
-Pre-sorting using magnetic beads before samples are run on the flow cytometer
-Analyze using a gene chip
10 minute, 30 minute, and 60 minute incubation periods are all similar
Full transcript