Affect of hypoxic conditions on mesenchymal stem cell

expansion and chondrogenic differentiation

Nikita Milani
University of Washington
Dr. Yasuyuki Sakai
Institute of Industrial Science

Abstract

As stem cells are grown and passaged they lose potency and their expansion rate is reduced. This makes it more
difficult to control their lineage during differentiation and greatly increases the cost of culturing large batches of stem
cells required for many types of research such as cartilage regeneration. There is evidence to suggest though that
culturing stem cells in more physiologically relevant conditions, such as in hypoxia, can increase the potency and
viability of mesenchymal stem cells. Here we show that culturing mesenchymal stem cells in hypoxic conditions on
oxygen-permeable polydimethylsiloxane (PDMS) instead of traditional non-permeable tissue culture polystyrene
(TCPS )yields better results for cell potency and viability for chondrogenic differentiation. These results are consistent
with our knowledge of in vivo conditions for MSCs and chondrocytes and results from related studies. Culturing
MSCs in improved conditions has the potential to greatly decrease the cost of MSC research and open the way to
new tissue engineering technologies.

Experiment 1: Expansion

Determine effect of substrate and oxygen

concentration on MSC expansion

Materials and methods:

Experiment 2: Differentiation
Aims:

Aims:

Process of collecting,
expanding, and using MSCs
for treatment

Materials and methods:

MSC stock (P6) thawed and cultured onto TCPS

and PDMS in MesenPRO medium
Separate plates stored in 20%, 10%, and 5%
oxygen incubators
Cells passaged every 3 days from P7 to P11

P11 cells centrifuged and cultured as

pellets in DMEM medium
Pellets stored in 20% oxygen incubator
Medium changed and collected for
metabolic analysis every 3 days for 14 days

Pellet culture

Results:

Results:
TCPS

Determine effect of substrate and oxygen

concentration on chondrogenic differentiation

PDMS

P8

20%

20%

10%

10%

5%
Figure 1: Images of MSCs
in expansion phase taken
on day 3

P11
P11
TCPS PDMS

5%
Figure 2: Graph comparing the
number of cells cultured in
different conditions at day 3

Figure 3: Images of pellet slices

stained with toluidine blue
Figure 4: Graphs showing the change in
metabolic markers over time

Conclusion:

Conclusion:

This experiment shows that substrate and oxygen

concentration strongly affect MSC expansion rate.
The optimal conditions found here are PDMS as the
substrate and 5% oxygen which most closely mimics
in vivo conditions for MSCs.

These results show that MSCs expanded under different

conditions have different viability during differentiation.
Toluidine blue stains for chondrogenic markers which are
seen most prevalently in pellets formed from MSCs cultured
on PDMS at 5% oxygen. The change in metabolism over
time shows that pellets formed from MSCs cultured on
PDMS and TCPS have differing metabolism. PDMS cultured
MSCs have a metabolism that most closely mimics
chondrocytes.

Perspective

References

This internship has been an incredible experience and has only confirmed
that my future plans are right for me. After earning my BS in
bioengineering in June 2016 I plan to go straight into graduate school to
pursue my Ph.D. I want to continue my research in the fields of stem cell
engineering, tissue engineering, and regenerative medicine. Working in
the lab of Dr. Sakai has proven to me that research is my passion and that
I will thrive in the graduate school environment.

Pattappa, G., HK Heywood, JD De Bruijn, and DA Lee. "The

Metabolism of Human Mesenchymal Stem Cells during Proliferation and

Differentiation." J Cell Physiol(2011)
Sato, M., M. Yamato, K. Hamahashi, T. Okano, and J. Mochida.
"Articular Cartilage Regeneration Using Cell Sheet Technology." The
Anatomical Record (2014)
Drela, K., A. Sarnowska, P. Siedlecka, I. Szablowska-Gadomska, B.
Lukomska, and K. Domanska-Janik. "Low Oxygen Atmosphere
Facilitates Proliferation and Maintains Undifferentiated State of
Umbilical Cordmesenchymal Stem Cells in an Hypoxia Inducible Factordependent Manner." Cytotherapy (2014)

TCPS

TCPS 2

PDMS

20%

10%

5%

Figure 1: Images of MSCs in expansion phase taken prior to tenth passage.

P8
20%

10%

5%

P11 TCPS

P11 PDMS

2cm

Title of Research Project:

Gothic 70pt

Name of student: Gothic 50pt

Affiliation of student: Gothic 50pt
Name of supervisor:

Photo of student

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Department of supervisor: Gothic 50pt

Abstract
Poster size A0 (80cm x 119cm)

Research Contents (Objectives or Aims, Materials &

Methods, Results & Discussions, Conclusions and
References)

2cm

2cm

Perspective

2cm

P8

P11
P11
TCPS PDMS

20%

10%

5%
Figure 3: Images of pellet slices
stained with toluidine blue