Cohen_Ben-Gurion University

Alginate Sponges
For 3-D Cell Culture
Smadar Cohen & Tsiona Elkayam
Ben-Gurion University of the Negev
Cohen_Ben-Gurion University
Alginate Sponge Characteristics
Physical Highly porous (>90%); Homogenous pore size
(100-200mm) and shape; Interconnecting pores
Transparent; Stable

Chemical
Polysaccharide (algae-derived) hydrogel, easily-
wetted by culture medium, allowing rapid and
efficient cell seeding and distribution
Biological Biocompatible with cells and the human body
Dissolution Mild conditions (EDTA, Citrate buffer)
Cell Recovery Easy, no cell damage,
maintaining surface cell markers (FACS analysis)
Shelf Life >12 months (room temperature, low humidity)
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Alginate Sponge
90% Porosity; ~200mm Pore Size
200mm
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Cell Distribution within
Alginate Sponge (10mm thick)
Surface
Bottom
Center
Initial cell seeding density
(1x10
8
cells/cm
3
)
H&E cross
sections
MTT
SEM
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3-D Culture in Alginate Sponge

Cell Source Cell Definition Source Cell Type
Isolation Primary, Adult Rat Hepatocytes- Pure Culture
Isolation Primary, Adult Rat Hepatocytes- Co-Culture with Kupffer
Cells or Non-Parenchymal Cells
Isolation Primary, Neonatal Rat Mixed hepatoblasts and oval (liver
stem) cells
ATCC (CRL-10741) Cell line, clone derivative of HepG2 Human Hepatocytes - C3A
Clonetics
(CC-2543)
Primary, Frozen Human Endothelial cells
dermal microvascular (HDMEC)
Isolation Primary, Fresh Human Endothelial cells
umbilical vein (HUVEC)
Gift
5


Cell line (transfected with encoding
SV-40 large T antigen)
Human Endothelial cells
dermal microvascular transfected
(HMEC-1)
Isolation Primary, Frozen Bovine Endothelial cells
bovine aortic (BAEC)
Clonetics (CC-2511) Primary, Frozen Human Dermal Fibroblast (NHDF)
Isolation Primary Rat Cardiac Fibroblasts
Isolation Primary, embryonic Rat Cardiac Myocytes
Isolation Primary, neonatal Rat Cardiac Myocytes
Technion (H-9-Wisconsin lines, Israeli lines) Human Embryonic stem cells
Isolation based on plate
adherence
Primary, Bone Marrow (BM) Human Mesenchymal stem cells
(MSC), BM
Isolation based on plate
adherence

Primary, Cord Blood (CB) Human MSC , CB (CD133+ enriched)
Isolation

Primary, CB Human Hematopoeitic stem cells
(HSC) , CB, CD133+ selection
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3-D Cell Applications
with Alginate Sponges
Tissue engineering
Bioproduction of valuable therapeutics
ADMETox research
Expansion of hematopoeitic and
mesenchymal stem cells
Stem cell differentiation
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Tissue Engineering of Cardiac Muscle
Schwarzkopf et al, Tissue Engineering, 12 (2006)
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Organization of Primary Hepatocytes
as Spheroids within Alginate Sponges
FDA staining SEM
TEM
Enhanced Hepatocellular Functions
Glicklis et al (2000, 2004); Dvir-Ginzberg et al (2003, 2004);
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The sponge recapitulates the stem cell niche
3-D microenvironment
The scaffold can be made cell specific by
attaching adhesion peptides (RGD, YIGSR)
The scaffold can be made a reservoir for
growth factors and molecular agents
important for cell growth and differentiation
High cell density can be reached in the
scaffold, without reaching confluence
Alginate Sponges for 3-D Stem Cells
Cohen_Ben-Gurion University
Release Rate
(ng/day
-1
)
K
A
binding to
M-Alginate (M
-1
)
Growth
Factor
24 6.32*10
6
VEGF
20 8.62*10
6
bFGF
7 2.82*10
7
aFGF
10 3.53*10
7
PDGF-
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Expansion and a better control over
differentiation of Human Embryonic
Stem (hES) Cells
Expansion of Hematopoietic stem
cells (HSC)
Expansion and induced differentiation
of Mesenchymal stem cells (MSC)


Stem Cell R&D
in Alginate Scaffold
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Human Embryonic Stem Cells (hESC)
Alginate sponge allows colonization of
undifferentiated hESC within pores
Alginate sponge pore size controls the
aggregation of human embryoid
bodies (hEB)
The alginate sponge maximizes hES
cell expansion and has a better
control over cell differentiation
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Morphology of hEBs
Scaffold-borne hEBs
Homogenous and Small Size
No Central Necrosis
Petri-Dish
Extensive Aggregation &
Central Necrosis
Gerecht-Nir et al, Biotechnol. Bioeng. (2004)
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Key Features of Sponge-borne hEBs
hEBs ranging between 250-900 mm
after 1 month, smaller than hEBs
formed in conventional Petri dishes
Spherical, homogenous in size relative
to those formed in Petri dishes
NO CENTRAL NECROSIS in hEBs
formed in alginate sponges
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Expansion of Human Embryonic Stem
Cells (hESC) in Alginate Sponges
4-Fold higher expansion in scaffold-borne EBs relative to Petri-dish
XTT
0
5
10
15
20
25
0 5 10 15 20 25 30
Day
C
e
l
l

(
1
0
<
6
>
/
m
l
)
Static
Scaf f olds
Petri Dish
Alginate Sponge
Seeding: 0.1-1 x10
5
/scaffold
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Differentiation within Alginate Borne-hEBs
into 3 Germ Layers
ECTODERM
MESODERM
ENDODERM

H&E
Neuronal cells (nestin+) Hepatocyte-like cells (a-FP+)

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Case Story:
Alginate vs. Collagen Scaffolds for ADMET

Collagen Alginate
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C3A- Clone Derivative of HepG2
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Collagen Alginate
C3A Cell Morphology in
Alginate vs. Collagen Scaffolds
3-D Spheroids
2-D Adherent Cells
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Laminin Expression in C3A seeded in
Alginate vs. Collagen Scaffolds
Collagen Alginate
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C3A Spheroids Express Albumin& CK18
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6b-testosterone (IU) Treatment
1.41 x 10
6

Cells only
302 x 10
6

Cells + Inducer
5.32 x 10
5

Cells + Inhibitor
6.0 x 10
5

Cells + Inducer +
Inhibitor
CYP3A4 Metabolism in C3A Spheroids
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nM Resorufin produced/
10
6
cells/1h
Treatment
C3A spheroids C3A cell
monolayer
2 0.7 0
Cells only
80 20 302
Cells + Inducer
0.60.6 0
Cells + Inhibitor
40 10 102
Cells + Inducer +
Inhibitor
CYP1A2 Metabolism in 2D vs. 3D Cultures
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1A2 Induction Alginate scaffold
0
5
10
15
20
25
A N.I A I
R
Q
1A2 Induction Collagen scaffold
0
2
4
6
8
10
12
14
16
C N.I C I
R
Q
1A2 Induction Alginate scaffold
0
5
10
15
20
25
A N.I A I
R
Q
1A2 Induction Collagen scaffold
0
2
4
6
8
10
12
14
16
C N.I C I
R
Q
1A2 mRNA levels in C3A seeded
in Alginate vs. Collagen Scaffolds
CYP1A2 Induction
Alginate scaffold Vs. Collagen Scaffold
0
20
40
60
80
100
120
140
C N.I A N.I C I A I
R
Q
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Summary- ADMET
3-D culture of C3A in alginate
scaffolds induces spheroid formation
Spheroids reveal high levels of
hepatocellular functions (protein
secretion, drug metabolism and
detoxification)
This construct is suitable for HTS
initial drug screening (ADMET)
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Summary- Why Alginate Sponges?

Ready to use for 3-D cell Culture
Algae-derived hydrogel
Optimized for specific cell types
Enhances cell activity
Degrades without changing local pH
Prolonged shelf life
Dimensionally stable in culture