Stem Cells Bioengineering 21th December 2012 Diana Santos nº 72459 MEBiom Sofia Sousa nº 54180 MEBiol
Jun 02, 2015
Stem Cells Bioengineering 21th December 2012
Diana Santos nº 72459 MEBiom
Sofia Sousa nº 54180 MEBiol
•Cellular densities similar to those in native tissues
•Diffusion limit of O2 and nutrients (Porosity and interconnectivity)
•Size, shape and material of the scaffolds
• Immune rejection in transplants
•Need for cellular expansion
“Regenerative Medicine is an interdisciplinary field of research
that applies the principles of engineering and the life sciences
towards the development of biological substitutes that
restore, maintain, or improve tissue function”
Langer & Vacanti
Tissue Engineering Limitations
hMSCs for Clinical Applications
•Graft-vs-Host disease treatment
•Bone grafts /Cartilage repair/Vertebral disks damage
•Coronary Heart Disease
•Parkinson’s, Alzheimer’s and epilepsy disease
• Incontinency/Renal failure/artificial bladder
•Burns
• Chron’s disease
• Myocardial ischemia
•Cornea/Retina substitution
•Cancer
• Important role in the co-transplant with HSC
Bladder Trachea
Intervertebral disk
Skin
Cornea
MSC Sources and Differentiation Process
Source: T.L. Bonfield, Discovery Medicine, 2010
T-Flask Spinner-flask Stirred Bioreactor Rotative Walls
TPS Roller Bottle Wave Bioreactor
Static Culture
•Non-homogeneous growth
•Non-homogeneous differentiation
•Low O2 and nutrients diffusion
•Difficulty of monitoring and control
•Low productivity
Dynamic Culture
•Better homogeneity
•O2 and nutrients supply during exposition to shear stress
•Higher cellular growth
•Higher control and productivity
Purpose
•Shear stress effect on osteoblastic differentiation of bioreactor culture beads
•Cellular position in a scaffold and it relation with cell proliferation
•Influence of radial position in hMSC osteoblastic differentiation
TPS bioreactor for 3D
dynamic culture of
hMSCs in spherical
alginate beads
Study
Source: Yeatts, A , Tissue Engineering, 2011
Landmark studies
Sikavitsas et al. (2003)
•Alginate -> support
proliferation and
osteoblastic
differentiation of BM
stromal cells
Mauney et al. (2005)
Utting et al. (2006)
• If low oxygen levels
are combined with
nutrient deprivation,
significant cell death
occurs (48h)
Potier, et al. (2007)
• Increased
proliferation and differentiation for hMSCs exposed to 2% O2 conditions compared to 20%
Grayson et al. (2007)
•Dextran does not
influence cell
differentiation and
proliferation
Li, et al. (2008)
Li, et al. (2009)
•Low oxygen (3%) concentrations can inhibit bone formation and in vitro osteoblastic differentiation
Iida et al. (2010)
Shear Stress and O2 Levels
Source: Yeatts, A , Tissue Engineering, 2011
Middle section of TPS growth chamber, 3mL/min flow rate
O2 concentrations throughout alginate beads Alginate bead diffussion model
cm/s
m
Flow velocities
•Higher in the contact points between beads
O2 concentration on the bead
•Static cultured falls to a minimum along the distance
•TPS minimum concentration in the center
1. Expansion in DMEM 10% FBS
2. Culture flasks (Passage each 3days)
3. Incubation at 37ºC, 5% CO2 (Passage each
6-7 days)
4. Osteogenic medium
hMSCs Culture
Calibration Curve: Outer annuli -> 18min
Alginate Beads and hMSCs Isolation
Experimental Groups
TPS large beads 4mm
TPS small beads 2mm
Control Groups
Static Culture large beads 4mm
Static culture small beads 2mm
Inner and outer annuli
Inner and outer annuli
Source: Yeatts, A , Tissue Engineering, 2011
5 mm
Sourc
e:
Bio
mate
rials
II,
IST,
2011
Alginate beads on static osteogenic media and TPS Bioreactor (3ml/min)
Bioreactor Design
Features
Incubator at 37ºC
Osteogenic media changed
every 3 days
1.0 mL/min for annuli studies
3.0 mL/min for shear stress studies
Growth Chamber
Platinum-cured silicone tubing dinner=6.4mm, douter=11.2mm, δ=2.4mm
High Permeability to O2 and CO2
Large δ -> Lower gas diffusion
Shear stress study
Experimental groups
TPS with 3% dextran
TPS with 9% dextran
Control Groups Static Culture
In 4mm beads
BMP-2
Day 1
Day 4
Day 8
Day 14
Day 21
Osteopontin (OPN)
Day 21 Day 14
Marker for osteoblastic
differentiation
Bone Morphogenetic Protein-2 and Osteopotin
BM
P-2
•Days 1,4,8 Weak correlation with shear stress
•Days 14,21 Strong correlation
OPN
•Shear stress increasing leads to higher OPN expression levels
•Day 21 shows higher [OPN] compared to day 14
Dependence of the expression levels of OPN
and BMP-2 with the shear stress
Shear stress study
For the same shear stress BMP-2 and OPN
levels are higher with each passing day
hMSCs Proliferation and Osteoblastic Differentation
in Relation to Position
Experimental
Groups
TPS large beads 4mm
TPS small beads 2mm
Control Groups
Static Culture large beads 4mm
Static culture small beads 2mm
hMSCs Proliferation in Relation to Position Pro
life
rati
on
•Day 1 -> all cells appeared viable
•Day 7 -> Increased proliferation in TPS small bead
•Day 14 –> Decreased proliferation in static large bead inner
•Day 21 –> Control beads have less proliferation compared to TPS beads
Live dead images of entire bead, inner annuli and small bead after one day
of bioreactor culture
Live and Dead Assay
1,000 μm
hMSCs Osteoblastic
Differentation
ALP
Day 1-14 -> ALP
expression is
higher in
controls
Day 21-> High
expression in TPS
and control, in
larger beads
inner annulli
OPN
Day 7-14 -> OPN
expressed low
levels
Day 21-> High
expression in TPS
larger beads
inner annulli and
small beads
ALP
Day 1
Day 7
Day 14
Day 21
OPN
Day 7
Day 14
Day 21
Mineralized matrix production
Day 7-14 -> Higher mineralization in
control small beads
Day 21-> Higher mineralization in TPS
inner annuli
hMSCs Osteoblastic Differentation
M
Day 1
Day 7
Day 14
Day 21
Proliferation
Differentiation Differentiation
Differentiation
Conclusions
Shear stress
Involved in temporal effect on the osteoblastic differentiation
High increase in OPN and BMP-2 in latest days
Osteoblastic differentiation
hMSCs position within scaffold plays a role in the osteoblastic differentiation of cells
MSCs may directed down a specific pathway by physical factors in their environment, helping the differentiation of inner cells of large beads
Oxygen levels and shear vary throughout the scaffold
Static culture of large beads leads to reduced osteoblastic differentiation and low mineralization
Proliferation
Dynamic culture can overcome the nutrients diffusion limitation in comparison to static culture
hMSCs position within scaffold play a role in the proliferation of cells
Bioreactor cultured small beads had the highest levels of proliferation
• Yeatts, Andrew B., et al (2012). “Human mesenchymal stem cell position within scaffolds
influences cell fate during dynamic culture” . Biotechnology and Bioengineering 109(9): 2381-
2391;
• Yeatts AB, Fisher JP. 2011b. “Tubular perfusion system for the long-term dynamic culture of
human mesenchymal stem cells”. Tissue Eng Part C Methods 17(3):337–348;
• Yeatts AB, et al (2012). “Bioreactors to influence stem cell fate: Augmentation of mesenchymal
stem cell signaling pathways via dynamic culture systems”. Biochimica et Biophysica Acta
• Salgado, A.J., O. P. Coutinho, et al (2004). “Bone and Tissue Engineerign: State of the Art and
Future Trends”. MacromolecularBioscience 4(8): 743-765
• Warren L. , et al (2007). “Hypoxia enhances proliferation and tissue formation of human
mesenchymal stem cells”. Biochemical and Biophysical Research 358 (3): 948 – 953;
• http://terpconnect.umd.edu/~jpfisher/index_files/presearch.htm
• Cell and Tissue Engineering – Biomaterials 2012 IST
• Biomaterials II – 2012 IST
• Stem Cell Bioengineerging – 2012 IST
References