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8/10/2019 ASCB-Alginate Sponge_SC 1206
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Cohen_Ben-Gurion University
Alginate Sponges
For 3-D Cell Culture
Smadar Cohen & Tsiona ElkayamBen-Gurion University of the Negev
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Alginate Sponge Characteristics
Physical Highly porous (>90%); Homogenous pore size
(100-200mm) and shape; Interconnecting pores
Transparent; Stable
ChemicalPolysaccharide (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 Sponge90% Porosity; ~200mm Pore Size
200mm
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Cell Distribution within
Alginate Sponge (10mm thick)
Surface
Bottom
Center
Initial cell seeding density
(1x108cells/cm3)
H&E cross
sections
MTT
SEM
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3-D Culture in Alginate Sponge
Cell SourceCell DefinitionSourceCell Type
IsolationPrimary, AdultRatHepatocytes- Pure Culture
IsolationPrimary, AdultRatHepatocytes- Co-Culture with KupfferCells or Non-Parenchymal Cells
IsolationPrimary, NeonatalRatMixed hepatoblasts and oval (liverstem) cells
ATCC (CRL-10741)Cell line, clone derivative of HepG2HumanHepatocytes - C3A
Clonetics(CC-2543)
Primary, FrozenHumanEndothelial cellsdermal microvascular (HDMEC)
IsolationPrimary, FreshHumanEndothelial cellsumbilical vein (HUVEC)
Gift5Cell line (transfected with encodingSV-40 large T antigen)
HumanEndothelial cellsdermal microvascular transfected(HMEC-1)
IsolationPrimary, FrozenBovineEndothelial cellsbovine aortic (BAEC)
Clonetics (CC-2511)Primary, FrozenHumanDermal Fibroblast (NHDF)
Isolation
Primary
Rat
Cardiac Fibroblasts
IsolationPrimary, embryonicRatCardiac Myocytes
IsolationPrimary, neonatalRatCardiac Myocytes
Technion(H-9-Wisconsin lines, Israeli lines)HumanEmbryonic stem cells
Isolation based on plateadherence
Primary, Bone Marrow (BM)HumanMesenchymal stem cells(MSC), BM
Isolation based on plateadherence
Primary, Cord Blood (CB)HumanMSC , CB (CD133+ enriched)
Isolation
Primary, CB
Human
Hematopoeitic stem cellsHSC CB CD133+ selection
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3-D Cell Applications
with Alginate Sponges
Tissue engineering
Bioproductionof valuable therapeutics
ADMEToxresearch
Expansionof hematopoeitic andmesenchymal 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 cellniche
3-D microenvironment
The scaffold can be madecell specific byattaching adhesion peptides(RGD, YIGSR)
The scaffold can be madea reservoirfor
growth factors and molecular agentsimportant for cell growth and differentiation
High cell density can be reachedin the
scaffold, without reaching confluence
Alginate Sponges for 3-D Stem Cells
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Release Rate
(ng/day-1)
KAbinding to
M-Alginate (M-1)
Growth
Factor
246.32*106VEGF
208.62*106bFGF
72.82*107aFGF
103.53*10
7
PDGF-
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Expansion and a better control over
differentiationof 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 colonizationof
undifferentiated hESC within pores
Alginate sponge pore sizecontrols theaggregationof human embryoid
bodies (hEB)
The alginate sponge maximizes hEScell expansionand 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, smallerthan hEBs
formed in conventional Petri dishes
Spherical, homogenous in sizerelative
to those formed in Petri dishes
NO CENTRAL NECROSISin 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
Cell(10/ml)
Static
Scaffolds
Petri Dish
Alginate Sponge
Seeding: 0.1-1 x105/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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C3A Cell Morphology in
Alginate vs. Collagen Scaffolds
3-D Spheroids2-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
1.41x 106
Cells only
302 x 106Cells + Inducer
5.32
x 105
Cells + Inhibitor
6.0 x 105
Cells + Inducer +
Inhibitor
CYP3A4 Metabolism in C3A Spheroids
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nM Resorufin produced/
106cells/1h
Treatment
C3A spheroidsC3A cellmonolayer
2 0.70Cells only
80 20302Cells + Inducer
0.60.60Cells + Inhibitor
40 10102
Cells + Inducer +
Inhibitor
CYP1A2 Metabolism in 2D vs. 3D Cultures
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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.IA N.IC IA I
R
Q
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Summary- ADMET
3-D culture of C3A in alginatescaffolds induces spheroidformation
Spheroids reveal high levels ofhepatocellular functions(proteinsecretion, drug metabolism anddetoxification)
This construct is suitable for HTSinitial drug screening (ADMET)
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Summary- Why Alginate Sponges?
Ready to use for 3-D cell Culture
Algae-derived hydrogelOptimized for specific cell types
Enhances cell activity
Degrades without changing local pHProlonged shelf life
Dimensionally stable in culture